Vaccines against coronavirus and methods of use

ABSTRACT

Disclosed herein are nucleic acid molecules encoding a SARS-CoV-2 spike antigen, SARS-CoV-2 spike antigens, immunogenic compositions, and vaccines and their use in inducing immune responses and protecting against or treating a Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infection in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 17/185,458,filed Feb. 25, 2021, which claims the benefit of U.S. Provisional Appl.No. 62/981,451, filed Feb. 25, 2020; U.S. Provisional Appl. No.63/004,380, filed Apr. 2, 2020; U.S. Provisional Appl. No. 63/028,404,filed May 21, 2020; U.S. Provisional Appl. No. 63/033,349, filed Jun. 2,2020; U.S. Provisional Appl. No. 63/040,865, filed Jun. 18, 2020; U.S.Provisional Appl. No. 63/046,415, filed Jun. 30, 2020; U.S. ProvisionalAppl. No. 63/062,762, filed Aug. 7, 2020; U.S. Provisional Appl. No.63/114,858, filed Nov. 17, 2020; U.S. Provisional Appl. No. 63/130,593filed Dec. 24, 2020; U.S. Provisional Appl. No. 63/136,973 filed Jan.13, 2021; U.S. Provisional Appl. No. 62/981,168, filed Feb. 25, 2020;U.S. Provisional Appl. No. 63/022,032, filed May 8, 2020; U.S.Provisional Appl. No. 63/056,996, filed Jul. 27, 2020; and U.S.Provisional Appl. No. 63/063,157, filed Aug. 7, 2020. The contents ofeach of these applications are incorporated herein by reference in theentirety.

TECHNICAL FIELD

The present invention relates to a vaccine for Severe Acute RespiratorySyndrome coronavirus 2 (SARS-CoV-2) and methods of administering thevaccine.

BACKGROUND

COVID-19, known previously as 2019-nCoV pneumonia or disease, hasrapidly emerged as a global threat to public health, joining severeacute respiratory syndrome (SARS) and Middle East respiratory syndrome(MERS) in a growing number of coronavirus-associated illnesses whichhave jumped from animals to people. There is an imminent need formedical countermeasures such as vaccines to combat the spread of suchemerging coronaviruses. There are at least seven identifiedcoronaviruses that infect humans, including MERS-CoV and SARS-CoV.

In December 2019, the city of Wuhan in China became the epicenter for aglobal outbreak of a novel coronavirus. This coronavirus, SARS-CoV-2,was isolated and sequenced from human airway epithelial cells frominfected patients (Zhu, et al. A Novel Coronavirus from Patients withPneumonia in China, 2019. N Engl J Med. 2020; Wu, et al. A newcoronavirus associated with human respiratory disease in China. Nature.2020). Disease symptoms can range from mild flu-like to severe caseswith life-threatening pneumonia (Huang, et al. Clinical features ofpatients infected with 2019 novel coronavirus in Wuhan, China. Lancet.2020). The global situation is dynamically evolving, and on Jan. 30,2020 the World Health Organization declared COVID-19 as a public healthemergency of international concern (PHEIC).

SEQUENCE LISTING

The instant application contains a Sequence Listing XML which is beingsubmitted herewith electronically in XML format and is herebyincorporated by reference in its entirety. The XML copy, created on May1, 2023, is named 104409000855_Sequence_Lising.xml and is 65,022 bytesin size.

SUMMARY

Provided herein are nucleic acid molecules encoding a SARS-CoV-2 spikeantigen. According to some embodiments, the encoded SARS-CoV-2 spikeantigen is a consensus antigen. In some embodiments, the nucleic acidmolecule comprises: a nucleic acid sequence having at least about 90%identity over an entire length of the nucleic acid sequence set forth innucleotides 55 to 3837 of SEQ ID NO: 2; a nucleic acid sequence havingat least about 90% identity over an entire length of SEQ ID NO: 2; thenucleic acid sequence of nucleotides 55 to 3837 of SEQ ID NO: 2; thenucleic acid sequence of SEQ ID NO: 2; a nucleic acid sequence having atleast about 90% identity over an entire length of SEQ ID NO: 3; thenucleic acid sequence of SEQ ID NO: 3; a nucleic acid sequence having atleast about 90% identity over an entire length of nucleotides 55 to 3837of SEQ ID NO: 5; a nucleic acid sequence having at least about 90%identity over an entire length of SEQ ID NO: 5; the nucleic acidsequence of nucleotides 55 to 3837 of SEQ ID NO: 5; the nucleic acidsequence of SEQ ID NO: 5; a nucleic acid sequence having at least about90% identity over an entire length of SEQ ID NO: 6; or the nucleic acidsequence of SEQ ID NO: 6. Also provided herein are nucleic acidmolecules encoding a SARS-CoV-2 spike antigen, wherein the SARS-CoV-2spike antigen comprises: an amino acid sequence having at least about90% identity over an entire length of residues 19 to 1279 of SEQ ID NO:1; the amino acid sequence set forth in residues 19 to 1279 of SEQ IDNO: 1; an amino acid sequence having at least about 90% identity over anentire length of SEQ ID NO: 1; the amino acid sequence of SEQ ID NO: 1;an amino acid sequence having at least about 90% identity over an entirelength of residues 19 to 1279 of SEQ ID NO: 4; an amino acid sequencehaving at least about 90% identity over an entire length of SEQ ID NO:4; the amino acid sequence set forth in residues 19 to 1279 of SEQ IDNO: 4, or the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the nucleic acid molecule encoding the SARS-CoV-2antigen is incorporated into a viral particle.

Further provided are vectors comprising the nucleic acid moleculeencoding the SARS-CoV-2 antigen. In some embodiments, the vector is anexpression vector. The nucleic acid molecule may be operably linked to aregulatory element selected from a promoter and a poly-adenylationsignal. The expression vector may be a plasmid or viral vector.

Immunogenic compositions comprising an effective amount of the vector orviral particle are disclosed. The immunogenic composition may comprise apharmaceutically acceptable excipient, such as but not limited to, abuffer. The buffer may optionally be saline-sodium citrate buffer. Insome embodiments, the immunogenic compositions comprise an adjuvant.

Also provided herein are SARS-CoV-2 spike antigens. According to someembodiments, the SARS-CoV-2 spike antigen is a consensus antigen. Insome embodiments, the SARS-CoV-2 spike antigen comprises: an amino acidsequence having at least about 90% identity over an entire length ofresidues 19 to 1279 of SEQ ID NO: 1; the amino acid sequence set forthin residues 19 to 1279 of SEQ ID NO: 1; an amino acid sequence having atleast about 90% identity over an entire length of SEQ ID NO: 1; theamino acid sequence of SEQ ID NO: 1; an amino acid sequence having atleast about 90% identity over an entire length of residues 19 to 1279 ofSEQ ID NO: 4; an amino acid sequence having at least about 90% identityover an entire length of SEQ ID NO: 4; the amino acid sequence set forthin residues 19 to 1279 of SEQ ID NO: 4; or the amino acid sequence ofSEQ ID NO: 4.

Further provided herein are vaccines for the prevention or treatment ofSevere Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infection.The vaccines comprising an effective amount of any one or combination ofthe aforementioned nucleic acid molecules, vectors, or antigens.According to some embodiments, the vaccine further comprises apharmaceutically acceptable excipient. The pharmaceutically acceptableexcipient may be a buffer, optionally saline-sodium citrate buffer.According to some embodiments, the vaccine further comprises anadjuvant.

Methods of inducing an immune response against Severe Acute RespiratorySyndrome coronavirus 2 (SARS-CoV-2) in a subject in need thereof arefurther provided. In come embodiments, the methods of inducing an immuneresponse comprise administering an effective amount of any one orcombination of the aforementioned nucleic acid molecules, vectors,immunogenic compositions, antigens, or vaccines to the subject. Alsoprovided herein are methods of protecting a subject in need thereof frominfection with SARS-CoV-2, the method comprising administering aneffective amount of any one or combination of the aforementioned nucleicacid molecules, vectors, immunogenic compositions, antigens, or vaccinesto the subject. Further provided are methods of treating SARS-CoV-2infection in a subject in need thereof, the method comprisingadministering an effective amount of any one or combination of theaforementioned nucleic acid molecules, vectors, immunogeniccompositions, antigens, or vaccines to the subject. In any of thesemethods, the administering may include at least one of electroporationand injection. According to some embodiments, the administeringcomprises parenteral administration, for example by intradermal,intramuscular, or subcutaneous injection, optionally followed byelectroporation. In some embodiments of the disclosed methods, aninitial dose of about 0.5 mg to about 2.0 mg of the nucleic acidmolecule is administered to the subject, optionally the initial dose is0.5 mg, 1.0 mg or 2.0 mg of the nucleic acid molecule. The methods mayfurther involve administration of a subsequent dose of about 0.5 mg toabout 2.0 mg of the nucleic acid molecule to the subject about fourweeks after the initial dose, optionally wherein the subsequent dose is0.5 mg, 1.0 mg or 2.0 mg of the nucleic acid molecule. In still furtherembodiments, the methods involve administration of one or more furthersubsequent doses of about 0.5 mg to about 2.0 mg of the nucleic acidmolecule to the subject at least twelve weeks after the initial dose,optionally wherein the further subsequent dose is 0.5 mg, 1.0 mg, or 2.0mg of the nucleic acid molecule. In any of these embodiments, INO-4800or a biosimilar thereof is administered.

Also provided herein are uses of any one or combination of the disclosednucleic acid molecules, vectors, immunogenic compositions, antigens, orvaccines in a method of inducing an immune response against Severe AcuteRespiratory Syndrome coronavirus 2 (SARS-CoV-2) in a subject in needthereof. Further provided are uses of any one or combination of thedisclosed nucleic acid molecules, vectors, immunogenic compositions,antigens, or vaccines in a method of protecting a subject from infectionwith Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). Alsoprovided herein are uses of any one or combination of the disclosednucleic acid molecules, vectors, immunogenic compositions, antigens, orvaccines in a method of treating a subject in need thereof againstSARS-CoV-2 infection. In accordance with any of these uses, the nucleicacid molecule, the vector, the immunogenic composition, the antigen, orthe vaccine may be administered to the subject by at least one ofelectroporation and injection. In some embodiments, the nucleic acidmolecule, the vector, the immunogenic composition, the antigen, or thevaccine is parenterally administered to the subject, for example byintradermal, intramuscular, or subcutaneous injection, optionallyfollowed by electroporation. In some embodiments of the disclosed uses,an initial dose of about 0.5 mg to about 2.0 mg of the nucleic acidmolecule is administered to the subject, optionally the initial dose is0.5 mg, 1.0 mg or 2.0 mg of the nucleic acid molecule. The uses mayfurther involve administration of a subsequent dose of about 0.5 mg toabout 2.0 mg of the nucleic acid molecule to the subject about fourweeks after the initial dose, optionally wherein the subsequent dose is0.5 mg, 1.0 mg or 2.0 mg of the nucleic acid molecule. In still furtherembodiments, the uses involve administration of one or more furthersubsequent doses of about 0.5 mg to about 2.0 mg of the nucleic acidmolecule to the subject at least twelve weeks after the initial dose,optionally wherein the further subsequent dose is 0.5 mg, 1.0 mg, or 2.0mg of the nucleic acid molecule. In any of these embodiments, INO-4800or a biosimilar thereof is administered.

Further provided herein are uses of any one or combination of thedisclosed nucleic acid molecules, vectors, immunogenic compositions,antigens, or vaccines in the preparation of a medicament. In someembodiments, the medicament is for treating or protecting againstinfection with Severe Acute Respiratory Syndrome coronavirus 2(SARS-CoV-2). In some embodiments, the medicament is for treating orprotecting against a disease or disorder associated with SARS-CoV-2infection. In some embodiments, the medicament is for treating orprotecting against Coronavirus Disease 2019 (COVID-19), Multisysteminflammatory syndrome in adults (MIS-A), or Multisystem inflammatorysyndrome in children (MIS-C).

The invention further relates to a method of detecting a persistentcellular immune response in a subject, the method comprising the stepsof: administering an immunogenic composition for inducing an immuneresponse against a SARS-CoV-2 antigen to a subject in need thereof;isolating peripheral mononuclear cells (PBMCs) from the subject;stimulating the isolated PBMCs with a spike antigen comprising an aminoacid sequence selected from the group consisting of: the amino acidsequence set forth in residues 19 to 1279 of SEQ ID NO: 1 the amino acidsequence of SEQ ID NO: 1; the amino acid sequence set forth in residues19 to 1279 of SEQ ID NO: 4; or the amino acid sequence of SEQ ID NO: 4;or a fragment thereof comprising at least 20 amino acids; and detectingat least one of the number of cytokine expressing cells and the level ofcytokine expression. In one embodiment, the step of detecting at leastone of the number of cytokine expressing cells and the level of cytokineexpression is performed using Enzyme-linked immunospot (ELISpot) orIntracellular Cytokine Staining (ICS) analysis using flow cytometry.

In one embodiment, the subject is administered an immunogeniccomposition comprising a nucleic acid molecule, wherein the nucleic acidmolecule comprises a nucleotide sequence encoding a peptide comprising:an amino acid sequence having at least about 90% identity over an entirelength of residues 19 to 1279 of SEQ ID NO: 1; the amino acid sequenceset forth in residues 19 to 1279 of SEQ ID NO: 1; an amino acid sequencehaving at least about 90% identity over an entire length of SEQ ID NO:1; the amino acid sequence of SEQ ID NO: 1; an amino acid sequencehaving at least about 90% identity over an entire length of residues 19to 1279 of SEQ ID NO: 4; an amino acid sequence having at least about90% identity over an entire length of SEQ ID NO: 4; the amino acidsequence set forth in residues 19 to 1279 of SEQ ID NO: 4; or the aminoacid sequence of SEQ ID NO: 4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, and 1D illustrate the design and expression ofSARS-CoV-2 synthetic DNA vaccine constructs. FIG. 1A shows a schematicdiagram of SARS-CoV-2 synthetic DNA vaccine constructs, pGX9501(matched) and pGX9503 (outlier (OL)) containing the IgE leader sequenceand SARS-CoV-2 spike protein insert (“Covid-19 spike antigen” or“Covid-19 spike-OL antigen”). FIG. 1B shows results of an RT-PCR assayof RNA extract from COS-7 cells transfected in duplicate with pGX9501 orpGX9503. Extracted RNA was analyzed by RT-PCR using PCR assays designedfor each target and for COS-7 β-Actin mRNA, used as an internalexpression normalization gene. Delta CT (Δ C_(T)) was calculated as theCT of the target minus the C_(T) of β-Actin for each transfectionconcentration and is plotted against the log of the mass of pDNAtransfected (Plotted as mean±SD). FIG. 1C shows analysis of in vitroexpression of Spike protein after transfection of 293T cells withpGX9501, pGX9503 or MOCK plasmid by Western blot. 293T cell lysates wereresolved on a gel and probed with a polyclonal anti-SARS Spike Protein.Blots were stripped then probed with an anti-β-actin loading control.FIG. 1D shows in vitro immunofluorescent staining of 293T cellstransfected with 3 μg/well of pGX9501, pGX9503 or pVax (empty controlvector). Expression of Spike protein was measured with polyclonalanti-SARS Spike Protein IgG and anti-IgG secondary. Cell nuclei werecounterstained with DAPI. Images were captured using IMAGEXPRESS™ Picoautomated cell imaging system.

FIG. 2 illustrates an IgG binding screen of a panel of SARS-CoV-2 andSARS-CoV antigens using sera from INO-4800-treated mice. BALB/c micewere immunized on Day 0 with 25 μg INO-4800 or pVAX-empty vector(Control) as described in the methods. Protein antigen binding of IgG at1:50 and 1:250 serum dilutions from mice at day 14. Data shown representmean OD450 nm values (mean+SD) for each group of 4 mice.

FIGS. 3A, 3B, 3C, and 3D demonstrate humoral responses to SARS-CoV-2 S1+2 and S receptor binding domain (RBD) protein antigen in BALB/c miceafter a single dose of INO-4800. BALB/c mice were immunized on Day 0with indicated doses of INO-4800 or pVAX-empty vector as described inExample 1. SARS-CoV-2 S1+2 (FIG. 3A) or SARS-CoV-2 RBD (FIG. 3B) proteinantigen binding of IgG in serial serum dilutions from mice at day 14 areshown. Data shown represent mean OD450 nm values (mean+SD) for eachgroup of 8 mice (FIGS. 3A and 3B) and 5 mice (FIGS. 3C and 3D). SerumIgG binding endpoint titers to SARS-CoV-2 S1+2 (FIG. 3B) and SARS-CoV-2RBD (FIG. 3D) protein. Data representative of 2 independent experiments.

FIGS. 4A and 4B illustrate neutralizing antibody responses afterimmunization with INO-4800. BALB/c mice (n of 5 per group) wereimmunized twice on days 0 and 14 with 10 μg of INO-4800. Sera wascollected on day 7 post-2nd immunization and serial dilutions wereincubated with a pseudovirus displaying the SARS-CoV-2 Spike andco-incubated with ACE2-293T cells. FIG. 4A shows neutralization ID50(mean±SD) in naïve and INO-4800 immunized mice. FIG. 4B shows relativeluminescence units (RLU) for sera from naive mice and mice vaccinatedwith INO-4800 as described in methods.

FIGS. 5A and 5B show humoral responses to SARS-CoV-2 in Hartley guineapigs after a single dose of INO-4800. Hartley guinea pigs mice wereimmunized on Day 0 with 100 μg INO-4800 or pVAX-empty vector asdescribed in Example 1. FIG. 5A shows SARS-CoV-2 S protein antigenbinding of IgG in serial serum dilutions at day 0 and 14. Data shownrepresent mean OD450 nm values (mean+SD) for the 5 guinea pigs. FIG. 5Bshows serum IgG binding titers (mean±SD) to SARS-CoV-2 S protein at day14. P values determined by Mann-Whitney test.

FIGS. 6A-6F demonstrate that INO-4800 immunized mouse and guinea pigsera compete with ACE2 receptor for SARS-CoV-2 Spike protein binding.FIG. 6A illustrates that soluble ACE2 receptor binds to CoV-2full-length spike with an EC50 of 0.025 μg/ml. FIG. 6B illustrates thatpurified serum IgG from BALB/c mice (n of 5 per group) after secondimmunization with INO-4800 yields significant competition against ACE2receptor. Serum IgG samples from the animals were run in triplicate.FIG. 6C illustrates that IgGs purified from n=5 mice day 7 post secondimmunization with INO-4800 show significant competition against ACE2receptor binding to SARS-CoV-2 S 1+2 protein. The soluble ACE2concentration for the competition assay is ˜0.1 μg/ml. Bars representthe mean and standard deviation of AUC. FIG. 6D illustrates Hartleyguinea pigs immunized on Day 0 and 14 with 100 μg INO-4800 or pVAX-emptyvector as described in the methods. Day 28 collected sera (diluted 1:20)was added to SARS-CoV-2 coated wells prior to the addition of serialdilutions of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 Sprotein was measured. Sera collected from 5 INO-4800-treated and 3pVAX-treated animals were used in this experiment. FIG. 6E illustratesserial dilutions of guinea pig sera collected on day 21 were added toSARS-CoV-2 coated wells prior to the addition of ACE2 protein. Detectionof ACE2 binding to SARS-CoV-2 S protein was measured. Sera collectedfrom 4 INO-4800-treated and 5 pVAX-treated guinea pigs were used in thisexperiment. FIG. 6F depicts IgGs purified from n=5 mice day 14 postsecond immunization with INO-4800 show competition against ACE2 receptorbinding to SARS-CoV-2 Spike protein compared to pooled naïve mice IgGs.Naïve mice were run in a single column. Vaccinated mice were run induplicate. If error bars are not visible, error is smaller than the datapoint.

FIGS. 7A-7D illustrate detection of SARS-CoV-2 S protein-reactiveantibodies in the BAL of INO-4800 immunized animals. BALB/c mice (n of 5per group) were immunized on days 0 and 14 with INO-4800 or pVAX and BALcollected at day 21 (FIGS. 7A and 7B). Hartley guinea pigs (n of 5 pergroup) were immunized on days 0, 14 and 21 with INO-4800 or pVAX and BALcollected at day 42 (FIGS. 7C and 7D). Bronchoalveolar lavage fluid wasassayed in duplicate for SARS-CoV-2 Spike protein-specific IgGantibodies by ELISA. Data are presented as endpoint titers (FIGS. 7A and7C), and BAL dilution curves with raw OD 450 nm values (FIGS. 7B and7D). In FIGS. 7A and 7C, bars represent the average of each group anderror bars the standard deviation. **p<0.01 by Mann-Whitney U test.

FIG. 8A-8C show induction of T cell responses in BALB/c micepost-administration of INO-4800. BALB/c mice (n=5/group) were immunizedwith 2.5 or 10 μg INO-4800. T cell responses were analyzed in theanimals on days 4, 7, 10 (FIGS. 8A and 8B), and day 14 (FIG. 8C). T cellresponses were measured by IFN-γ ELISpot in splenocytes stimulated for20 hours with overlapping peptide pools spanning the SARS-CoV-2 (FIG.8A), SARS-CoV (FIG. 8B), or MERS-CoV (FIG. 8C) Spike proteins. Barsrepresent the mean+SD.

FIGS. 9 and 10 illustrate cellular and humoral immune responses measuredin INO-4800-treated New Zealand White (NZW) rabbits. Day 0 and 28intradermal delivery of pDNA. PBMC IFN-γ ELISpot (FIG. 9 ); Serum IgGbinding ELISA (FIG. 10 ).

FIGS. 11A-11E illustrate humoral immune responses to SARS-CoV-2 spikeprotein measured in INO-4800 treated in rhesus monkeys. Day 0 and 28intradermal delivery of pDNA. Serum IgG binding ELISA.

FIGS. 12A-12G illustrate humoral immune responses to SARS and MERS spikeprotein measured in INO-4800 treated rhesus monkeys. Day 0 and 28intradermal delivery of pDNA. Serum IgG binding ELISA. (FIG. 12A-12G;left panel, 1 mg INO-4800; right panel, 2 mg INO-4800).

FIGS. 13A-13C illustrate cellular immune responses measured by PBMCIFN-γ ELISpot in INO-4800-treated in rhesus monkeys followingintradermal delivery of pDNA on days 0 and 28 intradermal. Results areshown in FIG. 13A (SARS CoV-2 Spike peptides); 13B (SARS CoV Spikepeptides); and 13C (MERS CoV Spike peptides).

FIGS. 14A and 14B show T cell epitope mapping after INO-4800administration to BALB/c mice. Splenocytes were stimulated for 20 hourswith SARS-CoV-2 peptide matrix mapping pools. FIG. 14A demonstrates Tcell responses following stimulation with matrix mapping SARS-CoV-2peptide pools. Bars represent the mean+SD of 5 mice. FIG. 14B shows themap of the SARS-CoV-2 Spike protein and identification of immunodominantpeptides in BALB/c mice. A known immunodominant SARS-CoV HLA-A2 epitopeis included for comparison. FIG. 14B discloses SEQ ID NOS 26-35,respectively, in order of appearance.

FIGS. 15A-15H depict humoral correlates of protection in throat andnasal compartments. (FIGS. 15A-15D) Correlation of throat viral load Log10 cDNA copies mL-1 at day 1 (FIGS. 15A, 15B) and day 3 (FIGS. 15C, 15D)post SARS-CoV-2 challenge with microneutralization titers (FIGS. 15A,15C) and RBD IgG binding endpoint titers (FIGS. 15B, 15D). (FIGS.15E-15H) Same analysis for nasal viral loads. P and R values providedfor two-sided non-parametric Spearman rank-correlation analyses. Controlanimals—red filled circles, INO-4800 X1—green filled circles andINO-4800 X2—blue filled circles.

FIG. 16 illustrates the Phase I study flow diagram.

FIGS. 17A, 17B, 17C, and 17D illustrate the humoral antibody response ofthe phase I clinical study. The humoral response in the 1.0 mg dosegroup and 2.0 mg dose group was assessed for the ability to neutralizeof live virus (n=18, 1.0 mg; n=19, 2.0 mg) (FIG. 17A); binding to theRBD regions (FIG. 17B); and binding to whole spike protein (51 and S2)(FIG. 17C). End point titers were calculated as the titer that exhibitedan OD 3.0 SD above baseline, titers at baseline were set at 1. In FIG.17D, the humoral response in the 1.0 mg dose group and 2.0 mg dose groupwas assessed for the ability to bind whole spike protein (51 and S2)(n=19, 1.0 mg; n=19, 2.0 mg). End point titers were calculated as thetiter that exhibited an OD 3.0 SD above baseline, titers at baselinewere set at 1. A response to live virus neutralization was a PRNTIC50≥10. In all graphs horizontal lines represent the Median and barsrepresent the Interquartile Range.

FIGS. 18A-18G illustrate Phase I clinical study cellular immune responseanalytical results. PBMCs isolated from vaccinated individuals werestimulated in vitro with SARS-CoV-2 spike antigen. The number of cellscapable of secreting IFN-gamma were measured in a standard ELISpot assayfor the 1.0 mg dose group and 2.0 mg dose group (FIG. 18A). Horizontallines represent Medians and bars represent Interquartile Ranges. Asshown in FIG. 18B, peptides spanning the entirety of the spike antigenwere divided into pools and tested individually in ELISpot, with poolsmapped to specific regions of the antigen. Three subjects are shownexemplifying the diversity of pool responses and associated magnitudeacross subjects. The pie chart represents the diversity of entirety ofthe 2.0 mg dose group. As illustrated in FIG. 18C, SARS-CoV-2 spikespecific cytokine production was measured from CD4+ and CD8+ T cells viaflow cytometry. Bars represent Mean response. Cytokine production isadditionally broken out in FIG. 18D using CCR7 and CD45RA into CentralMemory (CM), Effector Memory (EM) or Effector (E) differentiation statuswith data conveying what percentage of the overall cytokine responseoriginates from what differentiated group. Pie charts represent thepolyfunctionality of CD4+ and CD8+ T cells for each dose cohort areprovided in FIG. 18E. IL-4 production by CD4+ T cells for each dosecohort is illustrated in FIG. 18F. Horizontal lines represent Meanresponse. Graphs represent all evaluable subjects. Statistical analyseswere performed on all paired datasets. Those that were significant arenoted within the figure, lack of notation in the figure represents lackof statistical significance. FIG. 18G provides a heat map of eachsubject in the 2.0 mg dose group and the percentage of their ELISpotresponse dedicated to each pool covering the SARS-CoV-2 spike antigen.

FIG. 19A (post-first dose) and 19B (post-second dose) illustrate thePhase I Related Systemic and Local Adverse Events in severity of mild(Grade 1), moderate (Grade 2), severe (Grade 3) and life-threatening(Grade 4).

FIG. 20 provides supplementary data for humoral immune response. Threeconvalescent samples (all 3 with symptoms but non-hospitalized), testedby the ELISpot assay showed lower T cell responses, with a median of 33,than the 2.0 mg dose group at Week 8.

FIG. 21 provides supplementary Enzyme-linked immunospot (ELISpot) data.

FIGS. 22A-22F depict humoral and cellular responses in rhesus macaquesvaccinated with INO-4800. Study outline (FIG. 22A). Spike-specific IgG(FIG. 22B), RBD (FIG. 22C) and live virus-neutralising antibodies (FIG.22D) measured in serum from rhesus macaques that received 1 or 2 dosesof INO-4800 or were unvaccinated (Control). Lines represent thegeometric means. Cellular immune responses in rhesus macaques vaccinatedwith INO-4800. SARS-CoV-2 Spike-specific interferon gamma (IFNγ)secretion from PBMCs was measured in rhesus macaques that received 1 or2 doses of INO-4800 or were unvaccinated (Control) pre- (FIG. 22E) andpost-challenge (FIG. 22F). PBMCs were stimulated with 5 separate peptidepools spanning the spike protein and SFU frequencies measured inresponse to each pool summed. Lines represent the means.

FIGS. 23A-23C illustrate change in weight, temperature and hemoglobin inthe animals through the duration of the study. Animals received one(INO-4800X1) or two (INO-4800X2) doses of INO-4800 or were unvaccinated(control). Percentage change in body weights (FIG. 23A), temperature(FIG. 23B) and hemoglobin counts (FIG. 23C) of individual animals wererecorded and plotted at the indicated time points pre- andpost-challenge. Lines represent mean (FIG. 23A) and geometric mean (FIG.23B & FIG. 23C) value for each group.

FIGS. 24A-24F illustrate the upper respiratory tract viral loadsdetected by RT-qPCR following challenge with SARS-CoV-2. Animalsreceived one (INO-4800X1) or two (INO-4800X2) doses of INO-4800 or wereunvaccinated (control). Viral load plotted as Log 10 cDNA copies/ml foreach animal in throat swabs (FIGS. 24A-24C) and nasal swabs (FIGS.24D-24F). (FIGS. 24A&24D) Lines represent group geometric means with 95%CI. Area under the curve (AUC) of viral loads for throat swabs (FIG.24B) and nasal swabs (FIG. 24E) for each experimental group. Peak viralloads measured in each animal during the challenge period for throatswabs (FIG. 24C) and nasal swabs (FIG. 24F). LLOQ (lower limit ofquantification, 3.80 log copies/ml) and LLOD (lower limit of detection,3.47 log copies/ml). Positive samples detected below the LLOQ wereassigned the value of 3.80 log copies/ml. * p≤0.05 with Mann-Whitney ttest.

FIGS. 25A-25F illustrate the upper respiratory tract subgenomic viralloads detected by RT-qPCR following challenge with SARS-CoV-2. Animalsreceived one (INO-4800X1) or two (INO-4800X2) doses of INO-4800 or wereunvaccinated (control). Viral load plotted as Log 10 cDNA copies/ml foreach animal in throat swabs (FIGS. 25A-25C) and nasal swabs (FIGS.25D-25F). (FIGS. 25A and 25D) Lines represent group geometric means with95% CI. Area under the curve (AUC) of viral loads for throat swabs (FIG.25B) and nasal swabs (FIG. 25E) for each experimental group. Peak viralloads measured in each animal during the challenge period for throatswabs (FIG. 25C) and nasal swabs (FIG. 25F). LLOQ (4.11 log copies/mL)and LLOD (3.06 log copies/mL). Positive samples detected below the LLOQwere assigned the value of 3.81 log copies/ml.

FIGS. 26A-26D illustrate lower respiratory tract viral loads detected byRT-qPCR following challenge with SARS-CoV-2. Animals received one(INO-4800X1) or two (INO-4800X1) doses of INO-4800 or were unvaccinated(control). SARS-CoV-2 genomic and subgenomic viral loads were measuredfor individual animals in bronchoalveolar lavage (BAL (FIGS. 26A and26B)) and lung tissue (FIGS. 26C and 26D) samples collected at necropsy(6-8 days post challenge). Bars represent group medians. Assay LLOQ'sand LLOD's are provided in the methods section.

FIG. 27 illustrates viral RNA in animal tissue post challenge. Animalsreceived one (INO-4800X1) or two (INO-4800X2) doses of INO-4800 or wereunvaccinated (control). SARS-CoV-2 viral loads were measured forindividual animals in tissue samples collected at necropsy (6-8 DPC).Bars represent group median with 95% CI. Positive tissue samplesdetected below the limit of quantification (LoQ) of 4.76 log copies/mlwere assigned the value of 5 copies/μl, this equates to 4.46 logcopies/g, whilst undetected samples were assigned the value of <2.3copies/μl, equivalent to the assay's lower limit of detection (LoD)which equates to 4.76 log copies/g.

FIG. 28 shows representative histopathology (H&E stain) and presence ofSARS-CoV-2 viral RNA (ISH RNAScope stain) in animals vaccinated with 1dose (top), 2 doses (middle) or unvaccinated (bottom). Animalsvaccinated with 1 dose showed multifocal minimal to mild alveolar andinterstitial pneumonia (*), with higher severity in animal 10A. Theremaining animals from group 1 show minimal/mild inflammatoryinfiltrates (*). Mild perivascular cuffing was also observed(arrowheads) and viral RNA was shown by ISH within the lesions (arrows),abundantly in animal 10A, and in small amounts in animals 30A, 24A, 21Aand 38A (arrows). Animals vaccinated with 2 doses showed multifocalminimal to mild alveolar and interstitial pneumonia (*) together withminimal perivascular cuffing (arrowheads). Small quantities of viral RNAwere observed by ISH within the lesions from animals 9A, 45A, 33A and13A (arrows). Unvaccinated animals showed moderate multifocal alveolarand interstitial pneumonia (*), with presence of abundant viral RNAwithin the lesions from all animals (arrows).

FIGS. 29A-29G illustrate lung disease burden measured by histopathologyand CT scan following challenge with SARS-CoV-2. Total histopathologyscore (FIG. 29A), and image analysis of area positively stained area inISH RNAScope labelled sections for viral RNA (FIG. 29B). FIG. 29Cprovides a heat map illustration of histopathology scoring for eachparameter for individual animals. Total CT score (FIG. 29E). CTradiology scores for individual animals (FIGS. 29D-29G). FIG. 29D: Theextent of abnormality as a percentage of the lung affected. (FIG. 29E:COVID disease pattern with scoring based on presence of nodules, groundglass opacity, and consolidation. FIG. 29F: Zone classification (lung isdivided into 12 zones and each zone showing abnormalities is attributed1 point). FIG. 29G: Total cumulative CT score (Pattern+Zone scores).Line on graphs represent median value of group. * p≤0.05 withMann-Whitney t test.

FIG. 30 illustrates representative example of pulmonary abnormalitiesidentified on images constructed from CT scans. Images represent animalsthat did not receive a vaccination (control): 8A [A], 25A [B], 28A [C],14A [D], 50A [E]; animals that received a single dose of INO-4800vaccine: 10A [F], 21A [G], 38A [H]; animals that received two doses ofINO-4800 vaccine: 21A [I], 33A [J]. Arrows indicate areas of groundglass opacification and areas of consolidation. Images from macaquesthat did not have abnormal features are not shown.

FIG. 31A through FIG. 31F depict ELISpot images of IFN-γ+ mousesplenocytes after stimulation with SARS-CoV-2 and SARS antigens. Micewere immunized on day 0 and splenocytes harvested at the indicated timepoints. IFNγ-secreting cells in the spleens of immunized animals wereenumerated via ELISpot assay. Representative images show SARS-CoV-2specific (FIG. 31A through FIG. 31C) and SARS-CoV-specific (FIG. 31Dthrough FIG. 31F) IFNγ spot forming units in the splenocyte populationat days 4, 7, and 10 post-immunization. Images were captured byImmunoSpot CTL reader.

FIG. 32A and FIG. 32B depict flow cytometric analysis of T cellpopulations producing IFN-γ upon SARS-CoV-2 S protein stimulation.Splenocytes harvested from BALB/c and C57BL/6 mice 14 days after pVAX orINO-4800 treatment were made into single cell suspensions. The cellswere stimulated for 6 hours with SARS-CoV-2 overlapping peptide pools.FIG. 32A: CD4+ and CD8+ T cell gating strategy; singlets were gated on(i), then lymphocytes (ii) followed by live CD45+ cells (iii). Next CD3+cells were gated (iv) and from that population CD4+(v) and CD8+(vi)T-cells were gated. IFNγ+ cells were gated from each of the CD4+(vii)and CD8+(viii) T-cell populations. FIG. 32B: The percentage of CD4+ andCD8+ T cells producing IFNγ is depicted. Bars represent mean+SD. 4BALB/c and 4 C57BL/6 mice were used in this study. * p<0.05, MannWhitney test.

FIGS. 33A through 33H depict humoral and cellular immune responses inrhesus macaques. FIG. 33A: The study outline showing the vaccinationregimen and blood collection timepoints. FIG. 33B: Schematic ofSARS-CoV-2 spike protein. FIG. 33C: SARS-CoV-2 S1+S2 ECD, S1, RBD and S2protein antigen binding of IgG in serially diluted NHP sera collected onWeek 0, 2, 6, 12 and 15. Data represents the mean endpoint titers foreach individual NHP. (FIGS. 33D and 33E) Pseudoneutralization assayusing NHP sera, showing the presence of SARS-CoV-2 specific neutralizingantibodies against the D614 (FIG. 33D) and G614 (FIG. 33E) variants ofSARS-CoV-2. FIG. 33F and FIG. 33G: Serum collected at Week 6 fromINO-4800 vaccinated NHPs inhibited ACE2 binding. FIG. 33F: Plate-basedACE2 competition assay. FIG. 33G: Flow-based ACE2 inhibition assayshowing that inhibition of ACE2 binding in serially diluted NHP sera.FIG. 33H: T cell responses were measured by IFN-γ ELISpot in PBMCsharvested at weeks 0, 2, 6 and 15, and stimulated for 20 h withoverlapping peptide pools spanning the SARS-Cod′-2 Spike protein. Barsrepresent the mean+SD.

FIG. 34 depicts serum IgG cross-reactivity to SARS-CoV and MERS-CoVspike protein. IgG binding was measured in sera from INO-4800 vaccinatedrhesus macaques to SARS-CoV S1 and MERS-CoV S1 protein antigen.

FIG. 35 depicts bronchoalveolar lavage (BAL) IgG reactive to SARS-CoV-2S protein antigens. BAL samples collected from vaccinated animals wereassessed for SARS-CoV-2 reactive IgG binding to the full lengthSARS-CoV-2 spike protein and the RBD domain.

FIG. 36A and FIG. 36B depict exemplary experimental data demonstratingcellular response cross-reactivity to SARS-CoV and MERS-CoV spikeprotein. PBMC responses were analyzed by IFNγ ELISpot after stimulationwith overlapping peptide pools spanning the SARS-CoV-1 spike protein(FIG. 36A) and MERS-CoV spike protein (FIG. 36B). Bars represent themean+SD.

FIG. 37A through FIG. 37C depict exemplary experiments demonstratingrecall of humoral immune responses after viral challenge. FIG. 37A:Study outline. FIG. 37B: IgG binding ELISA. SARS-CoV-2 S1+S2 andSARS-CoV-2 RBD protein antigen binding of IgG in diluted NHP seracollected prior to challenge, during challenge and post challenge. FIG.37C: Pseudo-neutralization assay using NHP sera, showing the presence ofSARS-CoV-2 specific neutralizing antibodies against the D614 and G614variants of SARS-CoV-2 before and after viral challenge in INO-4800vaccinated (top panels) and naïve animals (bottom panels).

FIG. 38 depicts exemplary experiments demonstrating recall of cellularimmune responses after viral challenge. T cells responses were analyzedby IFNγ ELISpot in PBMCs stimulated with overlapping peptide poolsspanning the SARS-CoV-2 spike protein. Bars represent the mean+SD. Tcell responses analyzed by IFNγ ELISpot in PBMCs isolated pre and postchallenge with SARS-CoV-2 virus. Left panel naïve animals, right panelINO-4800 vaccinated animals.

FIG. 39 depicts exemplary experiments demonstrating recall of cellularimmune responses after viral challenge in individual rhesus macaques.Cellular responses were analyzed pre and post viral challenge by IFNγELISpot in PBMCs stimulated with overlapping peptide pools spanning theSARS-CoV-2 spike protein. Right panel naïve animals, left panel INO-4800vaccinated animals.

FIGS. 40A through 40F depict viral loads in the BAL fluid and Nasalswabs after viral challenge. At week 17 naïve and INO-4800 immunized (5per group) rhesus macaques were challenged by intranasal and intrachealadministration of 1.1×10⁴ PFU SARS-CoV-2 (US-WA1 isolate). FIG. 40A andFIG. 40D: Log sgmRNA copies/ml (FIG. 40A) in BAL (FIG. 40A), and NScopies/swab (FIG. 40D) were measured at multiple timepoints followingchallenge in naïve (left panels) and INO-4800 vaccinated (right panels)animals.

FIG. 40B and FIG. 40E: Peak viral loads (Between days 1 to 7) in BAL(FIG. 40B) and NS (FIG. 40E) following challenge. FIG. 40C and FIG. 40F:Viral RNA in BAL and NS at day 7 after challenge. Blue and Red linesreflect median viral loads. Mann-Whitney test P values are provided(FIG. 40B and FIG. 40C).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The term “comprising” is intended to include examples encompassed by theterms “consisting essentially of” and “consisting of”; similarly, theterm “consisting essentially of” is intended to include examplesencompassed by the term “consisting of” The present disclosure alsocontemplates other embodiments “comprising,” “consisting of” and“consisting essentially of” the embodiments or elements presentedherein, whether explicitly set forth or not.

It is to be appreciated that certain features of the disclosed materialsand methods which are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features of the disclosed materials andmethods that are, for brevity, described in the context of a singleembodiment, may also be provided separately or in any subcombination.

The singular forms “a,” “and” and “the” include plural references unlessthe context clearly dictates otherwise.

The term “about” when used in reference to numerical ranges, cutoffs, orspecific values is used to indicate that the recited values may vary byup to as much as 10% from the listed value. Thus, the term “about” isused to encompass variations of ±10% or less, variations of ±5% or less,variations of ±1% or less, variations of ±0.5% or less, or variations of±0.1% or less from the specified value. When values are expressed asapproximations by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. Reference to aparticular numerical value includes at least that particular valueunless the context clearly dictates otherwise.

“Adjuvant” as used herein means any molecule added to the vaccinedescribed herein to enhance the immunogenicity of the antigen.

“Antibody” as used herein means an antibody of classes IgG, IgM, IgA,IgD or IgE, or fragments, fragments or derivatives thereof, includingFab, F(ab′) 2, Fd, and single chain antibodies, diabodies, bispecificantibodies, bifunctional antibodies and derivatives thereof. Theantibody can be an antibody isolated from the serum sample of mammal, apolyclonal antibody, affinity purified antibody, or mixtures thereofwhich exhibits sufficient binding specificity to a desired epitope or asequence derived therefrom.

The term “biosimilar” (of an approved reference product/biological drug,i.e., reference listed drug) refers to a biological product that ishighly similar to the reference product notwithstanding minordifferences in clinically inactive components with no clinicallymeaningful differences between the biosimilar and the reference productin terms of safety, purity and potency, based upon data derived from (a)analytical studies that demonstrate that the biological product ishighly similar to the reference product notwithstanding minordifferences in clinically inactive components; (b) animal studies(including the assessment of toxicity); and/or (c) a clinical study orstudies (including the assessment of immunogenicity and pharmacokineticsor pharmacodynamics) that are sufficient to demonstrate safety, purity,and potency in one or more appropriate conditions of use for which thereference product is licensed and intended to be used and for whichlicensure is sought for the biosimilar. The biosimilar may be aninterchangeable product that may be substituted for the referenceproduct at the pharmacy without the intervention of the prescribinghealthcare professional. To meet the additional standard of“interchangeability,” the biosimilar is to be expected to produce thesame clinical result as the reference product in any given patient and,if the biosimilar is administered more than once to an individual, therisk in terms of safety or diminished efficacy of alternating orswitching between the use of the biosimilar and the reference product isnot greater than the risk of using the reference product without suchalternation or switch. The biosimilar utilizes the same mechanisms ofaction for the proposed conditions of use to the extent the mechanismsare known for the reference product. The condition or conditions of useprescribed, recommended, or suggested in the labeling proposed for thebiosimilar have been previously approved for the reference product. Theroute of administration, the dosage form, and/or the strength of thebiosimilar are the same as those of the reference product and thebiosimilar is manufactured, processed, packed or held in a facility thatmeets standards designed to assure that the biosimilar continues to besafe, pure and potent. The biosimilar may include minor modifications inthe amino acid sequence when compared to the reference product, such asN- or C-terminal truncations that are not expected to change thebiosimilar performance.

“Coding sequence” or “encoding nucleic acid” as used herein means thenucleic acids (RNA or DNA molecule) that comprise a nucleotide sequencewhich encodes a protein. The coding sequence can further includeinitiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of an individual or mammal to whichthe nucleic acid is administered.

“Complement” or “complementary” as used herein means Watson-Crick (e.g.,A-T/U and C-G) or Hoogsteen base pairing between nucleotides ornucleotide analogs of nucleic acid molecules.

“Consensus” or “Consensus Sequence” as used herein may mean a syntheticnucleic acid sequence, or corresponding polypeptide sequence,constructed based on analysis of an alignment of multiple subtypes of aparticular antigen. The sequence may be used to induce broad immunityagainst multiple subtypes, serotypes, or strains of a particularantigen. Synthetic antigens, such as fusion proteins, may be manipulatedto generate consensus sequences (or consensus antigens).

“Electroporation,” “electro-permeabilization,” or “electro-kineticenhancement” (“EP”) as used interchangeably herein means the use of atransmembrane electric field pulse to induce microscopic pathways(pores) in a bio-membrane; their presence allows biomolecules such asplasmids, oligonucleotides, siRNA, drugs, ions, and water to pass fromone side of the cellular membrane to the other.

“Fragment” as used herein means a nucleic acid sequence or a portionthereof that encodes a polypeptide capable of eliciting an immuneresponse in a mammal. The fragments can be DNA fragments selected fromat least one of the various nucleotide sequences that encode proteinfragments set forth below.

“Fragment” or “immunogenic fragment” with respect to polypeptidesequences means a polypeptide capable of eliciting an immune response ina mammal that cross reacts with a full-length wild type strainSARS-CoV-2 antigen. Fragments of consensus proteins can comprise atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% or at least 95% of aconsensus protein. In some embodiments, fragments of consensus proteinscan comprise at least 20 amino acids or more, at least 30 amino acids ormore, at least 40 amino acids or more, at least 50 amino acids or more,at least 60 amino acids or more, at least 70 amino acids or more, atleast 80 amino acids or more, at least 90 amino acids or more, at least100 amino acids or more, at least 110 amino acids or more, at least 120amino acids or more, at least 130 amino acids or more, at least 140amino acids or more, at least 150 amino acids or more, at least 160amino acids or more, at least 170 amino acids or more, at least 180amino acids or more, at least 190 amino acids or more, at least 200amino acids or more, at least 210 amino acids or more, at least 220amino acids or more, at least 230 amino acids or more, or at least 240amino acids or more of a consensus protein.

As used herein, the term “genetic construct” refers to the DNA or RNAmolecules that comprise a nucleotide sequence which encodes a protein.The coding sequence includes initiation and termination signals operablylinked to regulatory elements including a promoter and polyadenylationsignal capable of directing expression in the cells of the individual towhom the nucleic acid molecule is administered. As used herein, the term“expressible form” refers to gene constructs that contain the necessaryregulatory elements operable linked to a coding sequence that encodes aprotein such that when present in the cell of the individual, the codingsequence will be expressed.

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences, means that the sequences have aspecified percentage of residues that are the same over a specifiedregion. The percentage can be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) can be considered equivalent.Identity can be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

“Immune response” as used herein means the activation of a host's immunesystem, e.g., that of a mammal, in response to the introduction ofantigen. The immune response can be in the form of a cellular or humoralresponse, or both.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or “nucleic acidmolecule” as used herein means at least two nucleotides covalentlylinked together. The depiction of a single strand also defines thesequence of the complementary strand. Thus, a nucleic acid alsoencompasses the complementary strand of a depicted single strand. Manyvariants of a nucleic acid can be used for the same purpose as a givennucleic acid. Thus, a nucleic acid also encompasses substantiallyidentical nucleic acids and complements thereof. A single strandprovides a probe that can hybridize to a target sequence under stringenthybridization conditions. Thus, a nucleic acid also encompasses a probethat hybridizes under stringent hybridization conditions.

Nucleic acids can be single stranded or double-stranded or can containportions of both double-stranded and single-stranded sequence. Thenucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid can contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids can be obtained by chemical synthesismethods or by recombinant methods.

“Operably linked” as used herein means that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter can be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene can beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance can be accommodated withoutloss of promoter function.

A “peptide,” “protein,” or “polypeptide” as used herein can mean alinked sequence of amino acids and can be natural, synthetic, or amodification or combination of natural and synthetic.

“Promoter” as used herein means a synthetic or naturally derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter can comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter can also comprise distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A promoter can bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter can regulate the expression of a genecomponent constitutively or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, and CMV IE promoter.

“Signal peptide” and “leader sequence” are used interchangeably hereinand refer to an amino acid sequence that can be linked at the aminoterminus of a SARS-CoV-2 protein set forth herein. Signalpeptides/leader sequences typically direct localization of a protein.Signal peptides/leader sequences used herein preferably facilitatesecretion of the protein from the cell in which it is produced. Signalpeptides/leader sequences are often cleaved from the remainder of theprotein, often referred to as the mature protein, upon secretion fromthe cell. Signal peptides/leader sequences are linked at the N terminusof the protein.

“Subject” as used herein can mean a mammal that wants or is in need ofbeing immunized with a herein described immunogenic composition orvaccine. The mammal can be a human, chimpanzee, guinea pig, dog, cat,horse, cow, mouse, rabbit, or rat.

“Substantially identical” as used herein can mean that a first andsecond amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100 or more amino acids. Substantiallyidentical can also mean that a first nucleic acid sequence and a secondnucleic acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100 or more nucleotides.

“Treatment” or “treating,” as used herein can mean protecting of ananimal from a disease through means of preventing, suppressing,repressing, or completely eliminating the disease. Preventing thedisease involves administering an immunogenic composition or a vaccineof the present invention to an animal prior to onset of the disease.Suppressing the disease involves administering an immunogeniccomposition or a vaccine of the present invention to an animal afterinduction of the disease but before its clinical appearance. Repressingthe disease involves administering an immunogenic composition or avaccine of the present invention to an animal after clinical appearanceof the disease.

As used herein, unless otherwise noted, the term “clinically proven”(used independently or to modify the terms “safe” and/or “effective”)shall mean that it has been proven by a clinical trial wherein theclinical trial has met the approval standards of U.S. Food and DrugAdministration, EMA or a corresponding national regulatory agency. Forexample, proof may be provided by the clinical trial(s) described in theexamples provided herein.

The term “clinically proven safe”, as it relates to a dose, dosageregimen, treatment or method with a SARS-CoV-2 antigen (for example, aSARS-CoV-2 spike antigen administered as pGX9501 or INO-4800 or abiosimilar thereof) refers to a favorable risk:benefit ratio with anacceptable frequency and/or acceptable severity of treatment-emergentadverse events (referred to as AEs or TEAEs) compared to the standard ofcare or to another comparator. An adverse event is an untoward medicaloccurrence in a patient administered a medicinal product. One index ofsafety is the National Cancer Institute (NCI) incidence of adverseevents (AE) graded per Common Toxicity Criteria for Adverse Events CTCAEv4.03.

The terms “clinically proven efficacy” and “clinically proven effective”as used herein in the context of a dose, dosage regimen, treatment ormethod refer to the effectiveness of a particular dose, dosage ortreatment regimen. Efficacy can be measured based on change in thecourse of the disease in response to an agent of the present invention.For example, a SARS-CoV-2 antigen (for example, a SARS-CoV-2 spikeantigen administered as pGX9501 or INO-4800 or a biosimilar thereof) isadministered to a patient in an amount and for a time sufficient toinduce an improvement, preferably a sustained improvement, in at leastone indicator that reflects the severity of the disorder that is beingtreated. Various indicators that reflect the extent of the subject'sillness, disease or condition may be assessed for determining whetherthe amount and time of the treatment is sufficient. Such indicatorsinclude, for example, clinically recognized indicators of diseaseseverity, symptoms, or manifestations of the disorder in question. Thedegree of improvement generally is determined by a physician, who maymake this determination based on signs, symptoms, biopsies, or othertest results, and who may also employ questionnaires that areadministered to the subject, such as quality-of-life questionnairesdeveloped for a given disease. For example, a SARS-CoV-2 antigen (forexample, a SARS-CoV-2 spike antigen administered as pGX9501 or INO-4800or a biosimilar thereof) may be administered to achieve an improvementin a patient's condition related to SARS-CoV-2 infection. Improvementmay be indicated by an improvement in an index of disease activity, byamelioration of clinical symptoms or by any other measure of diseaseactivity.

“Variant” used herein with respect to a nucleic acid means (i) a portionor fragment of a referenced nucleotide sequence; (ii) the complement ofa referenced nucleotide sequence or portion thereof; (iii) a nucleicacid that is substantially identical to a referenced nucleic acid or thecomplement thereof; or (iv) a nucleic acid that hybridizes understringent conditions to the referenced nucleic acid, complement thereof,or a sequences substantially identical thereto.

Variant can further be defined as a peptide or polypeptide that differsin amino acid sequence by the insertion, deletion, or conservativesubstitution of amino acids, but retain at least one biologicalactivity. Representative examples of “biological activity” include theability to be bound by a specific antibody or to promote an immuneresponse. Variant can also mean a protein with an amino acid sequencethat is substantially identical to a referenced protein with an aminoacid sequence that retains at least one biological activity. Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity, degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157:105-132 (1982). The hydropathic index of an amino acid is based on aconsideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes can be substituted andstill retain protein function. In one aspect, amino acids havinghydropathic indexes of ±2 are substituted. The hydrophilicity of aminoacids can also be used to reveal substitutions that would result inproteins retaining biological function. A consideration of thehydrophilicity of amino acids in the context of a peptide permitscalculation of the greatest local average hydrophilicity of thatpeptide, a useful measure that has been reported to correlate well withantigenicity and immunogenicity. Substitution of amino acids havingsimilar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions can be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

A variant may be a nucleic acid sequence that is substantially identicalover the full length of the full gene sequence or a fragment thereof.The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical over the full length of the gene sequence or a fragmentthereof. A variant may be an amino acid sequence that is substantiallyidentical over the full length of the amino acid sequence or fragmentthereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical over the full length of the amino acid sequence or afragment thereof.

“Vector” as used herein means a nucleic acid sequence containing anorigin of replication. A vector can be a viral vector, bacteriophage,bacterial artificial chromosome or yeast artificial chromosome. A vectorcan be a DNA or RNA vector. A vector can be a self-replicatingextrachromosomal vector, and preferably, is a DNA plasmid.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

Nucleic Acid Molecules, Antigens, and Immunogenic Compositions

Provided herein are immunogenic compositions, such as vaccines,comprising a nucleic acid molecule encoding a SARS-CoV-2 antigen, afragment thereof, a variant thereof, or a combination thereof. Alsoprovided herein are immunogenic compositions, such as vaccines,comprising a SARS-CoV-2 antigen, a fragment thereof, a variant thereof,or a combination thereof. The immunogenic compositions can be used toprotect against and treat any number of strains of SARS-CoV-2, therebytreating, preventing, and/or protecting against SARS-CoV-2-basedpathologies. The immunogenic compositions can significantly induce animmune response of a subject administered the immunogenic compositions,thereby protecting against and treating SARS-CoV-2 infection.

The immunogenic composition can be a DNA vaccine, a peptide vaccine, ora combination DNA and peptide vaccine. The DNA vaccine can include anucleic acid molecule encoding the SARS-CoV-2 antigen. The nucleic acidmolecule can be DNA, RNA, cDNA, a variant thereof, a fragment thereof,or a combination thereof. The nucleic acid molecule can also includeadditional sequences that encode linker, leader, or tag sequences thatare linked to the nucleic acid molecule encoding the SARS-CoV-2 antigenby a peptide bond. The peptide vaccine can include a SARS-CoV-2antigenic peptide, a SARS-CoV-2 antigenic protein, a variant thereof, afragment thereof, or a combination thereof. The combination DNA andpeptide vaccine can include the above described nucleic acid moleculeencoding the SARS-CoV-2 antigen and the SARS-CoV-2 antigenic peptide orprotein, in which the SARS-CoV-2 antigenic peptide or protein and theencoded SARS-CoV-2 antigen have the same amino acid sequence.

The disclosed immunogenic compositions can elicit both humoral andcellular immune responses that target the SARS-CoV-2 antigen in thesubject administered the immunogenic composition. The disclosedimmunogenic compositions can elicit neutralizing antibodies andimmunoglobulin G (IgG) antibodies that are reactive with the SARS-CoV-2spike antigen. The immunogenic composition can also elicit CD8+ and CD4+T cell responses that are reactive to the SARS-CoV-2 antigen and produceinterferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), andinterleukin-2 (IL-2).

The immunogenic composition can induce a humoral immune response in thesubject administered the immunogenic composition. The induced humoralimmune response can be specific for the SARS-CoV-2 antigen. The inducedhumoral immune response can be reactive with the SARS-CoV-2 antigen. Thehumoral immune response can be induced in the subject administered thevaccine by about 1.5-fold to about 16-fold, about 2-fold to about12-fold, or about 3-fold to about 10-fold. The humoral immune responsecan be induced in the subject administered the vaccine by at least about1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at leastabout 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, atleast about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold,at least about 6.0-fold, at least about 6.5-fold, at least about7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at leastabout 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, atleast about 10.0-fold, at least about 10.5-fold, at least about11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at leastabout 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, atleast about 14.0-fold, at least about 14.5-fold, at least about15.0-fold, at least about 15.5-fold, or at least about 16.0-fold.

The humoral immune response induced by the immunogenic composition caninclude an increased level of neutralizing antibodies associated withthe subject administered the immunogenic composition as compared to asubject not administered the immunogenic composition. The neutralizingantibodies can be specific for the SARS-CoV-2 antigen. The neutralizingantibodies can be reactive with the SARS-CoV-2 antigen. The neutralizingantibodies can provide protection against and/or treatment of SARS-CoV-2infection and its associated pathologies in the subject administered theimmunogenic composition.

The humoral immune response induced by the immunogenic composition caninclude an increased level of IgG antibodies associated with the subjectadministered the immunogenic composition as compared to a subject notadministered the immunogenic composition. These IgG antibodies can bespecific for the SARS-CoV-2 antigen. These IgG antibodies can bereactive with the SARS-CoV-2 antigen. The level of IgG antibodyassociated with the subject administered the immunogenic composition canbe increased by about 1.5-fold to about 16-fold, about 2-fold to about12-fold, or about 3-fold to about 10-fold as compared to the subject notadministered the immunogenic composition. The level of IgG antibodyassociated with the subject administered the immunogenic composition canbe increased by at least about 1.5-fold, at least about 2.0-fold, atleast about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold,at least about 4.0-fold, at least about 4.5-fold, at least about5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at leastabout 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, atleast about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold,at least about 9.5-fold, at least about 10.0-fold, at least about10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at leastabout 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, atleast about 13.5-fold, at least about 14.0-fold, at least about14.5-fold, at least about 15.0-fold, at least about 15.5-fold, or atleast about 16.0-fold as compared to the subject not administered theimmunogenic composition.

The immunogenic composition can induce a cellular immune response in thesubject administered the immunogenic composition. The induced cellularimmune response can be specific for the SARS-CoV-2 antigen. The inducedcellular immune response can be reactive to the SARS-CoV-2 antigen. Theinduced cellular immune response can include eliciting a CD8+ T cellresponse. The elicited CD8+ T cell response can be reactive with theSARS-CoV-2 antigen. The elicited CD8+ T cell response can bepolyfunctional. The induced cellular immune response can includeeliciting a CD8+ T cell response, in which the CD8+ T cells produceinterferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α),interleukin-2 (IL-2), or a combination of IFN-γ and TNF-α.

The induced cellular immune response can include an increased CD8+ Tcell response associated with the subject administered the immunogeniccomposition as compared to the subject not administered the immunogeniccomposition. The CD8+ T cell response associated with the subjectadministered the immunogenic composition can be increased by about2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-foldto about 20-fold as compared to the subject not administered theimmunogenic composition. The CD8+ T cell response associated with thesubject administered the immunogenic composition can be increased by atleast about 1.5-fold, at least about 2.0-fold, at least about 3.0-fold,at least about 4.0-fold, at least about 5.0-fold, at least about6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at leastabout 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, atleast about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold,at least about 10.5-fold, at least about 11.0-fold, at least about11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at leastabout 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, atleast about 14.5-fold, at least about 15.0-fold, at least about16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at leastabout 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, atleast about 22.0-fold, at least about 23.0-fold, at least about24.0-fold, at least about 25.0-fold, at least about 26.0-fold, at leastabout 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, orat least about 30.0-fold as compared to the subject not administered theimmunogenic composition.

The induced cellular immune response can include an increased frequencyof CD3+CD8+ T cells that produce IFN-γ. The frequency of CD3+CD8+IFN-γ+T cells associated with the subject administered the immunogeniccomposition can be increased by at least about 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or20-fold as compared to the subject not administered the immunogeniccomposition.

The induced cellular immune response can include an increased frequencyof CD3+CD8+ T cells that produce TNF-α. The frequency of CD3+CD8+TNF-α+T cells associated with the subject administered the immunogeniccomposition can be increased by at least about 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-fold, or 14-fold as compared to the subject not administered theimmunogenic composition.

The induced cellular immune response can include an increased frequencyof CD3+CD8+ T cells that produce IL-2. The frequency of CD3+CD8+IL-2+ Tcells associated with the subject administered the immunogeniccomposition can be increased by at least about 0.5-fold, 1.0-fold,1.5-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, or5.0-fold as compared to the subject not administered the immunogeniccomposition.

The induced cellular immune response can include an increased frequencyof CD3+CD8+ T cells that produce both IFN-γ and TNF-α. The frequency ofCD3+CD8+IFN-γ+ TNF-α+ T cells associated with the subject administeredthe immunogenic composition can be increased by at least about 25-fold,30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold,70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold,110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, or180-fold as compared to the subject not administered the immunogeniccomposition.

The cellular immune response induced by the immunogenic composition caninclude eliciting a CD4+ T cell response. The elicited CD4+ T cellresponse can be reactive with the SARS-CoV-2 antigen. The elicited CD4+T cell response can be polyfunctional. The induced cellular immuneresponse can include eliciting a CD4+ T cell response, in which the CD4+T cells produce IFN-γ, TNF-α, IL-2, or a combination of IFN-γ and TNF-α.

The induced cellular immune response can include an increased frequencyof CD3+CD4+ T cells that produce IFN-γ. The frequency of CD3+CD4+IFN-γ+T cells associated with the subject administered the immunogeniccomposition can be increased by at least about 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or20-fold as compared to the subject not administered the immunogeniccomposition.

The induced cellular immune response can include an increased frequencyof CD3+CD4+ T cells that produce TNF-α. The frequency of CD3+CD4+ TNF-α+T cells associated with the subject administered the immunogeniccomposition can be increased by at least about 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold,21-fold, or 22-fold as compared to the subject not administered theimmunogenic composition.

The induced cellular immune response can include an increased frequencyof CD3+CD4+ T cells that produce IL-2. The frequency of CD3+CD4+IL-2+ Tcells associated with the subject administered the immunogeniccomposition can be increased by at least about 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold,21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold,29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold,37-fold, 38-fold, 39-fold, 40-fold, 45-fold, 50-fold, 55-fold, or60-fold as compared to the subject not administered the immunogeniccomposition.

The induced cellular immune response can include an increased frequencyof CD3+CD4+ T cells that produce both IFN-γ and TNF-α. The frequency ofCD3+CD4+IFN-γ+ TNF-α+ associated with the subject administered theimmunogenic composition can be increased by at least about 2-fold,2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold,6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold,9.5-fold, 10.0-fold, 10.5-fold, 11.0-fold, 11.5-fold, 12.0-fold,12.5-fold, 13.0-fold, 13.5-fold, 14.0-fold, 14.5-fold, 15.0-fold,15.5-fold, 16.0-fold, 16.5-fold, 17.0-fold, 17.5-fold, 18.0-fold,18.5-fold, 19.0-fold, 19.5-fold, 20.0-fold, 21-fold, 22-fold, 23-fold24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold,32-fold, 33-fold, 34-fold, or 35-fold as compared to the subject notadministered the immunogenic composition.

The immunogenic composition of the present invention can have featuresrequired of effective immunogenic compositions such as being safe so theimmunogenic composition itself does not cause illness or death; isprotective against illness resulting from exposure to live pathogenssuch as viruses or bacteria; induces neutralizing antibody to preventinvention of cells; induces protective T cells against intracellularpathogens; and provides ease of administration, few side effects,biological stability, and low cost per dose.

The immunogenic composition can further induce an immune response whenadministered to different tissues such as the muscle or skin. Theimmunogenic composition can further induce an immune response whenadministered via electroporation, or injection, or subcutaneously, orintramuscularly.

a. SARS-CoV-2 Antigen and Nucleic Acid Molecules Encoding the Same

As described above, provided herein are immunogenic compositionscomprising a nucleic acid molecule encoding a SARS-CoV-2 antigen, afragment thereof, a variant thereof, or a combination thereof. Alsoprovided herein are immunogenic compositions comprising a SARS-CoV-2antigen, a fragment thereof, a variant thereof, or a combinationthereof.

Upon binding cell surface proteins and membrane fusion, the coronavirusenters the cell and its singled-stranded RNA genome is released into thecytoplasm of the infected cell. The singled-stranded RNA genome is apositive strand and thus, can be translated into a RNA polymerase, whichproduces additional viral RNAs that are minus strands. Accordingly, theSARS-CoV-2 antigen can also be a SARS-CoV-2 RNA polymerase.

The viral minus RNA strands are transcribed into smaller, subgenomicpositive RNA strands, which are used to translate other viral proteins,for example, nucleocapsid (N) protein, envelope (E) protein, and matrix(M) protein. Accordingly, the SARS-CoV-2 antigen can comprise aSARS-CoV-2 nucleocapsid protein, a SARS-CoV-2 envelope protein, aSARS-CoV-2 matrix protein, or a fragment of the S1 subunit comprisingthe SARS-CoV-2 Spike Receptor Binding Domain (RBD).

The viral minus RNA strands can also be used to replicate the viralgenome, which is bound by nucleocapsid protein. Matrix protein, alongwith spike protein, is integrated into the endoplasmic reticulum of theinfected cell. Together, the nucleocapsid protein bound to the viralgenome and the membrane-embedded matrix and spike proteins are buddedinto the lumen of the endoplasmic reticulum, thereby encasing the viralgenome in a membrane. The viral progeny are then transported by golgivesicles to the cell membrane of the infected cell and released into theextracellular space by endocytosis.

Coronaviruses, including SARS-CoV-2, are encapsulated by a membrane andhave a type 1 membrane glycoprotein known as spike (S) protein, whichforms protruding spikes on the surface of the coronavirus. TheSARS-CoV-2 S protein is a class I membrane fusion protein, which is themajor envelope protein on the surface of coronaviruses. The spikeprotein facilitates binding of the coronavirus to proteins located onthe surface of a cell, for example, the metalloprotease amino peptidaseN, and mediates cell-viral membrane fusion. In particular, the spikeprotein contains an S1 subunit that facilitates binding of thecoronavirus to cell surface proteins. Accordingly, the S1 subunit of thespike protein controls which cells are infected by the coronavirus. Thespike protein also contains a S2 subunit, which is a transmembranesubunit that facilitates viral and cellular membrane fusion.Accordingly, the SARS-CoV-2 antigen can comprise a SARS-CoV-2 spikeprotein, a S1 subunit of a SARS-CoV-2 spike protein, or a S2 subunit ofa SARS-CoV-2 spike protein.

In some embodiments, the SARS-CoV-2 antigen can be a SARS-CoV-2 spikeprotein, a SARS-CoV-2 RNA polymerase, a SARS-CoV-2 nucleocapsid protein,a SARS-CoV-2 envelope protein, a SARS-CoV-2 matrix protein, a fragmentthereof, a variant thereof, or a combination thereof.

The SARS-CoV-2 antigen can be a SARS-CoV-2 spike antigen, a fragmentthereof, a variant thereof, or a combination thereof. The SARS-CoV-2spike antigen is capable of eliciting an immune response in a mammalagainst one or more SARS-CoV-2 strains. The SARS-CoV-2 spike antigen cancomprise an epitope(s) that makes it particularly effective as animmunogen against which an anti-SARS-CoV-2 immune response can beinduced.

The SARS-CoV-2 antigen can be a consensus antigen derived from two ormore strains of SARS-CoV-2. In some embodiments, the SARS-CoV-2 antigenis a SARS-CoV-2 consensus spike antigen. The SARS-CoV-2 consensus spikeantigen can be derived from the sequences of spike antigens from strainsof SARS-CoV-2, and thus, the SARS-CoV-2 consensus spike antigen isunique. In some embodiments, the SARS-CoV-2 consensus spike antigen canbe an outlier spike antigen, having a greater amino acid sequencedivergence from other SARS-CoV-2 spike proteins. Accordingly, theimmunogenic compositions of the present invention are widely applicableto multiple strains of SARS-CoV-2 because of the unique sequences of theSARS-CoV-2 consensus spike antigen. These unique sequences allow thevaccine to be universally protective against multiple strains ofSARS-CoV-2, including genetically diverse variants of SARS-CoV-2.Nucleic acid molecules encoding the SARS-CoV-2 antigen can be modifiedfor improved expression. Modification can include codon optimization,RNA optimization, addition of a kozak sequence for increased translationinitiation, and/or the addition of an immunoglobulin leader sequence toincrease the immunogenicity of the SARS-CoV-2 antigen. The SARS-CoV-2spike antigen can comprise a signal peptide such as an immunoglobulinsignal peptide, for example, but not limited to, an immunoglobulin E(IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, theSARS-CoV-2 spike antigen can comprise a hemagglutinin (HA) tag. TheSARS-CoV-2 spike antigen can be designed to elicit stronger and broadercellular and/or humoral immune responses than a corresponding codonoptimized spike antigen.

In some embodiments, the SARS-CoV-2 antigen comprises an amino acidsequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity over an entire length of residues 19 to 1279 of SEQ ID NO: 1.In some embodiments the SARS-CoV-2 antigen comprises the amino acidsequence set forth in residues 19 to 1279 of SEQ ID NO: 1. In someembodiments, the SARS-CoV-2 antigen comprises an amino acid sequencehaving at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity overan entire length of SEQ ID NO: 1. In some embodiments the SARS-CoV-2antigen comprises the amino acid sequence of SEQ ID NO: 1. In someembodiments the nucleic acid molecule encoding the SARS-CoV-2 antigencomprises the nucleotide sequence having at least about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identity to the sequence set forth in nucleotides55 to 3837 of SEQ ID NO:2, SEQ ID NO: 2, or SEQ ID NO: 3.

In some embodiments the SARS-CoV-2 antigen comprises an amino acidsequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity over an entire length of residues 19 to 1279 of SEQ ID NO: 4 orover an entire length of SEQ ID NO: 4. In some embodiments theSARS-CoV-2 antigen comprises the amino acid sequence set forth inresidues 19 to 1279 of SEQ ID NO: 4. In some embodiments the SARS-CoV-2antigen comprises the amino acid sequence of SEQ ID NO: 4. In someembodiments the nucleic acid molecule encoding the SARS-CoV-2 antigencomprises: a nucleic acid sequence having at least about 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identity over an entire length of nucleotides 55to 3837 of SEQ ID NO: 5 or over an entire length of SEQ ID NO: 5; thenucleic acid sequence of nucleotides 55 to 3837 of SEQ ID NO: 5; thenucleic acid sequence of SEQ ID NO: 5; a nucleic acid sequence having atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over an entirelength of SEQ ID NO: 6; or the nucleic acid sequence of SEQ ID NO: 6.

In some embodiments the SARS-CoV-2 antigen is operably linked to an IgEleader sequence. In some such embodiments, the SARS-CoV-2 antigencomprises the amino acid sequence set forth in SEQ ID NO: 1. In someembodiments, the SARS-CoV-2 antigen is encoded by the nucleotidesequence set forth in SEQ ID NO:2 or SEQ ID NO: 3. In some embodimentsin which the SARS-CoV-2 antigen includes an IgE leader, the SARS-CoV-2antigen comprises the amino acid sequence set forth in SEQ ID NO: 4. Insome such embodiments, the SARS-CoV-2 antigen is encoded by thenucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO: 6.

Immunogenic fragments of SEQ ID NO:1 can be provided. Immunogenicfragments can comprise at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO:1. Insome embodiments, immunogenic fragments include a leader sequence, suchas for example an immunoglobulin leader, such as the IgE leader. In someembodiments, immunogenic fragments are free of a leader sequence.

Immunogenic fragments of proteins with amino acid sequences homologousto immunogenic fragments of SEQ ID NO:1 can be provided. Suchimmunogenic fragments can comprise at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% ofproteins that are 95% homologous to SEQ ID NO:1. Some embodiments relateto immunogenic fragments that have 96% homology to the immunogenicfragments of consensus protein sequences herein. Some embodiments relateto immunogenic fragments that have 97% homology to the immunogenicfragments of consensus protein sequences herein. Some embodiments relateto immunogenic fragments that have 98% homology to the immunogenicfragments of consensus protein sequences herein. Some embodiments relateto immunogenic fragments that have 99% homology to the immunogenicfragments of consensus protein sequences herein. In some embodiments,immunogenic fragments include a leader sequence, such as for example animmunoglobulin leader, such as the IgE leader. In some embodiments,immunogenic fragments are free of a leader sequence.

Some embodiments relate to immunogenic fragments of SEQ ID NO:1.Immunogenic fragments can be at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO:1.Immunogenic fragments can be at least 95%, at least 96%, at least 97% atleast 98% or at least 99% homologous to fragments of SEQ ID NO:1. Insome embodiments, immunogenic fragments include sequences that encode aleader sequence, such as for example an immunoglobulin leader, such asthe IgE leader. In some embodiments, fragments are free of codingsequences that encode a leader sequence.

Immunogenic fragments of SEQ ID NO:4 can be provided. Immunogenicfragments can comprise at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO:4. Insome embodiments, immunogenic fragments include a leader sequence, suchas for example an immunoglobulin leader, such as the IgE leader. In someembodiments, immunogenic fragments are free of a leader sequence.

Immunogenic fragments of proteins with amino acid sequences homologousto immunogenic fragments of SEQ ID NO:4 can be provided. Suchimmunogenic fragments can comprise at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% ofproteins that are 95% homologous to SEQ ID NO:4. Some embodiments relateto immunogenic fragments that have 96% homology to the immunogenicfragments of consensus protein sequences herein. Some embodiments relateto immunogenic fragments that have 97% homology to the immunogenicfragments of consensus protein sequences herein. Some embodiments relateto immunogenic fragments that have 98% homology to the immunogenicfragments of consensus protein sequences herein. Some embodiments relateto immunogenic fragments that have 99% homology to the immunogenicfragments of consensus protein sequences herein. In some embodiments,immunogenic fragments include a leader sequence, such as for example animmunoglobulin leader, such as the IgE leader. In some embodiments,immunogenic fragments are free of a leader sequence.

Some embodiments relate to immunogenic fragments of SEQ ID NO:4.Immunogenic fragments can be at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO:4.Immunogenic fragments can be at least 95%, at least 96%, at least 97% atleast 98% or at least 99% homologous to fragments of SEQ ID NO:4. Insome embodiments, immunogenic fragments include sequences that encode aleader sequence, such as for example an immunoglobulin leader, such asthe IgE leader. In some embodiments, fragments are free of codingsequences that encode a leader sequence.

b. Vector

The immunogenic compositions can comprise one or more vectors thatinclude a nucleic acid molecule encoding the SARS-CoV-2 antigen. The oneor more vectors can be capable of expressing the antigen. The vector canhave a nucleic acid sequence containing an origin of replication. Thevector can be a plasmid, bacteriophage, bacterial artificial chromosomeor yeast artificial chromosome. The vector can be either aself-replicating extrachromosomal vector or a vector which integratesinto a host genome.

The one or more vectors can be an expression construct, which isgenerally a plasmid that is used to introduce a specific gene into atarget cell. Once the expression vector is inside the cell, the proteinthat is encoded by the gene is produced by the cellular-transcriptionand translation machinery ribosomal complexes. The plasmid is frequentlyengineered to contain regulatory sequences that act as enhancer andpromoter regions and lead to efficient transcription of the gene carriedon the expression vector. The vectors of the present invention expresslarge amounts of stable messenger RNA, and therefore proteins.

The vectors may have expression signals such as a strong promoter, astrong termination codon, adjustment of the distance between thepromoter and the cloned gene, and the insertion of a transcriptiontermination sequence and a PTIS (portable translation initiationsequence).

(1) Expression Vectors

The vector can be a circular plasmid or a linear nucleic acid. Thecircular plasmid and linear nucleic acid are capable of directingexpression of a particular nucleotide sequence in an appropriate subjectcell. The vector can have a promoter operably linked to theantigen-encoding nucleotide sequence, which may be operably linked totermination signals. The vector can also contain sequences required forproper translation of the nucleotide sequence. The vector comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components. The expression of the nucleotide sequence in theexpression cassette may be under the control of a constitutive promoteror of an inducible promoter, which initiates transcription only when thehost cell is exposed to some particular external stimulus. In the caseof a multicellular organism, the promoter can also be specific to aparticular tissue or organ or stage of development.

(2) Circular and Linear Vectors

The vector may be a circular plasmid, which may transform a target cellby integration into the cellular genome or exist extrachromosomally(e.g., autonomous replicating plasmid with an origin of replication).

The vector can be pVAX, pcDNA3.0, pGX-0001, or provax, or any otherexpression vector capable of expressing DNA encoding the antigen andenabling a cell to translate the sequence to an antigen that isrecognized by the immune system.

Also provided herein is a linear nucleic acid immunogenic composition,or linear expression cassette (“LEC”), that is capable of beingefficiently delivered to a subject via electroporation and expressingone or more desired antigens. The LEC may be any linear DNA devoid ofany phosphate backbone. The DNA may encode one or more antigens. The LECmay contain a promoter, an intron, a stop codon, and/or apolyadenylation signal. The expression of the antigen may be controlledby the promoter. The LEC may not contain any antibiotic resistance genesand/or a phosphate backbone. The LEC may not contain other nucleic acidsequences unrelated to the desired antigen gene expression.

The LEC may be derived from any plasmid capable of being linearized. Theplasmid may be capable of expressing the antigen. The plasmid can be pNP(Puerto Rico/34) or pM2 (New Caledonia/99). The plasmid may be WLV009,pVAX, pcDNA3.0, or provax, or any other expression vector capable ofexpressing DNA encoding the antigen and enabling a cell to translate thesequence to an antigen that is recognized by the immune system.

The LEC can be perM2. The LEC can be perNP. perNP and perMR can bederived from pNP (Puerto Rico/34) and pM2 (New Caledonia/99),respectively.

(3) Promoter, Intron, Stop Codon, and Polyadenylation Signal

The vector may have a promoter. A promoter may be any promoter that iscapable of driving gene expression and regulating expression of theisolated nucleic acid. Such a promoter is a cis-acting sequence elementrequired for transcription via a DNA dependent RNA polymerase, whichtranscribes the antigen sequence described herein. Selection of thepromoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter may be positionedabout the same distance from the transcription start in the vector as itis from the transcription start site in its natural setting. However,variation in this distance may be accommodated without loss of promoterfunction.

The promoter may be operably linked to the nucleic acid sequenceencoding the antigen and signals required for efficient polyadenylationof the transcript, ribosome binding sites, and translation termination.The promoter may be a CMV promoter, SV40 early promoter, SV40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or another promotershown effective for expression in eukaryotic cells.

The vector may include an enhancer and an intron with functional splicedonor and acceptor sites. The vector may contain a transcriptiontermination region downstream of the structural gene to provide forefficient termination. The termination region may be obtained from thesame gene as the promoter sequence or may be obtained from differentgenes.

c. Excipients and Other Components of the Immunogenic Compositions

The immunogenic compositions may further comprise a pharmaceuticallyacceptable excipient. The pharmaceutically acceptable excipient can befunctional molecules such as vehicles, carriers, buffers, or diluents.As used herein. “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Thebuffer generally has a pH from about 4.0 to about 8.0, for example fromabout 5.0 to about 7.0. In some embodiments, the buffer is saline-sodiumcitrate (SSC) buffer. In some embodiments in which the immunogeniccomposition comprises a nucleic acid molecule encoding a SARS-CoV-2spike antigen as described above, the immunogenic composition comprises10 mg/ml of vector in buffer, for example but not limited to SSC buffer.In some embodiments, the immunogenic composition comprises 10 mg/mL ofthe DNA plasmid pGX9501 or pGX9503 in buffer. In some embodiments, theimmunogenic composition is stored at about 2° C. to about 8° C. In someembodiments, the immunogenic composition is stored at room temperature.The immunogenic composition may be stored for at least a year at roomtemperature. In some embodiments, the immunogenic composition is stableat room temperature for at least a year, wherein stability is defined asa supercoiled plasmid percentage of at least about 80%. In someembodiments, the supercoiled plasmid percentage is at least about 85%following storage for at least a year at room temperature.

The pharmaceutically acceptable excipient can be a transfectionfacilitating agent, which can include surface active agents, such asimmune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPSanalog including monophosphoryl lipid A, muramyl peptides, quinoneanalogs, vesicles such as squalene and squalene, hyaluronic acid,lipids, liposomes, calcium ions, viral proteins, polyanions,polycations, or nanoparticles, or other known transfection facilitatingagents.

The transfection facilitating agent may be a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. The transfectionfacilitating agent is poly-L-glutamate, and the poly-L-glutamate may bepresent in the immunogenic composition at a concentration less than 6mg/ml. The transfection facilitating agent may also include surfaceactive agents such as immune-stimulating complexes (ISCOMS), Freundsincomplete adjuvant, LPS analog including monophosphoryl lipid A,muramyl peptides, quinone analogs and vesicles such as squalene andsqualene, and hyaluronic acid may also be used administered inconjunction with the genetic construct. The DNA plasmid immunogeniccompositions may also include a transfection facilitating agent such aslipids, liposomes, including lecithin liposomes or other liposomes knownin the art, as a DNA-liposome mixture (see for example WO9324640),calcium ions, viral proteins, polyanions, polycations, or nanoparticles,or other known transfection facilitating agents. The transfectionfacilitating agent is a polyanion, polycation, includingpoly-L-glutamate (LGS), or lipid. Concentration of the transfectionagent in the immunogenic composition is less than 4 mg/ml, less than 2mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml,less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, orless than 0.010 mg/ml.

The pharmaceutically acceptable excipient can be an adjuvant. Theadjuvant can be other genes that are expressed in an alternative plasmidor are delivered as proteins in combination with the plasmid above inthe immunogenic composition. The adjuvant may be selected from the groupconsisting of: α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon,platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermalgrowth factor (EGF), cutaneous T cell-attracting chemokine (CTACK),epithelial thymus-expressed chemokine (TECK), mucosae-associatedepithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 includingIL-15 having the signal sequence deleted and optionally including thesignal peptide from IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK,TECK, platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF,epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10,IL-12, IL-18, or a combination thereof.

Other genes that can be useful as adjuvants include those encoding:MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin,CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1,ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18,CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7,IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNFreceptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF,DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1,Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K,SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND,NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 andfunctional fragments thereof.

The immunogenic composition may further comprise a genetic vaccinefacilitator agent as described in U.S. Ser. No. 021,579 filed Apr. 1,1994, which is fully incorporated by reference.

The immunogenic composition can be formulated according to the mode ofadministration to be used. According to some embodiments, theimmunogenic composition is formulated in a buffer, optionallysaline-sodium citrate buffer. For example, the immunogenic compositionmay formulated at a concentration of 10 mg nucleic acid molecule permilliliter of a sodium salt citrate buffer. An injectable immunogenicpharmaceutical composition can be sterile, pyrogen free and particulatefree. An isotonic formulation or solution can be used. Additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitol,and lactose. The immunogenic composition can comprise a vasoconstrictionagent. The isotonic solutions can include phosphate buffered saline.Immunogenic compositions can further comprise stabilizers includinggelatin and albumin. The stabilizers can allow the formulation to bestable at room or ambient temperature for extended periods of time,including LGS or polycations or polyanions.

Also provided herein are articles of manufacture comprising theimmunogenic composition. In some embodiments, the article of manufactureis a container holding the immunogenic composition. The container maybe, for example but not limited to, a syringe or a vial. The vial mayhave a stopper piercable by a syringe.

The immunogenic composition can be packaged in suitably sterilizedcontainers such as ampules, bottles, or vials, either in multi-dose orin unit dosage forms. The containers are preferably hermetically sealedafter being filled with a vaccine preparation. Preferably, the vaccinesare packaged in a container having a label affixed thereto, which labelidentifies the vaccine, and bears a notice in a form prescribed by agovernment agency such as the United States Food and Drug Administrationreflecting approval of the vaccine under appropriate laws, dosageinformation, and the like. The label preferably contains informationabout the vaccine that is useful to a health care professionaladministering the vaccine to a patient. The package also preferablycontains printed informational materials relating to the administrationof the vaccine, instructions, indications, and any necessary requiredwarnings.

Methods of Vaccination

Also provided herein are methods of treating, protecting against, and/orpreventing disease in a subject in need thereof by administering theimmunogenic composition to the subject. Administration of theimmunogenic composition to the subject can induce or elicit an immuneresponse in the subject. The induced immune response can be used totreat, prevent, and/or protect against disease, for example, pathologiesrelating to SARS-CoV-2 infection. The induced immune response in thesubject administered the immunogenic composition can provide resistanceto one or more SARS-CoV-2 strains.

The induced immune response can include an induced humoral immuneresponse and/or an induced cellular immune response. The humoral immuneresponse can be induced by about 1.5-fold to about 16-fold, about 2-foldto about 12-fold, or about 3-fold to about 10-fold. The induced humoralimmune response can include IgG antibodies and/or neutralizingantibodies that are reactive to the antigen. The induced cellular immuneresponse can include a CD8+ T cell response, which is induced by about2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-foldto about 20-fold.

The vaccine dose can be between 1 μg to 10 mg active component/kg bodyweight/time, and can be 20 μg to 10 mg component/kg body weight/time.The vaccine can be administered every 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more days or every 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moreweeks. The number of vaccine doses for effective treatment can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more.

In one embodiment, the total vaccine dose is 1.0 mg of nucleic acid. Inone embodiment, the total vaccine dose is 2.0 mg of nucleic acid,administered as 2×1.0 mg nucleic acid.

a. Administration

The immunogenic composition can be formulated in accordance withstandard techniques well known to those skilled in the pharmaceuticalart. Such compositions can be administered in dosages and by techniqueswell known to those skilled in the medical arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular subject, and the route of administration. The vaccine may beadministered, for example, in one, two, three, four, or more injections.In some embodiments, an initial dose of about 0.5 mg to about 2.0 mg ofthe nucleic acid molecule is administered to the subject. The initialdose may be administered in one, two, three, or more injections. Theinitial dose may be followed by administration of one, two, three, four,or more subsequent doses of about 0.5 mg to about 2.0 mg of the nucleicacid molecule about one, two, three, four, five, six, seven, eight, ten,twelve or more weeks after the immediately prior dose. Each subsequentdose may be administered in one, two, three, or more injections. In someembodiments, the immunogenic composition is administered to the subjectbefore, with, or after the additional agent. In some embodiments, theimmunogenic composition is administered as a booster followingadministration of an agent for the treatment of SARS-CoV-2 infection orthe treatment or prevention of a disease or disorder associated withSARS-CoV-2 infection. In one embodiment, the disease or disorderassociated with SARS-CoV-2 infection includes, but is not limited to,Coronavirus Disease 2019 (COVID-19) and/or Multisystem inflammatorysyndrome in adults (MIS-A) or Multisystem inflammatory syndrome inchildren (MIS-C).

The subject can be a mammal, such as a human, a horse, a nonhumanprimate, a cow, a pig, a sheep, a cat, a dog, a guinea pig, a rabbit, arat, or a mouse.

The vaccine can be administered prophylactically or therapeutically. Inprophylactic administration, the vaccines can be administered in anamount sufficient to induce an immune response. In therapeuticapplications, the vaccines are administered to a subject in need thereofin an amount sufficient to elicit a therapeutic effect. An amountadequate to accomplish this is defined as “therapeutically effectivedose.” Amounts effective for this use will depend on, e.g., theparticular composition of the vaccine regimen administered, the mannerof administration, the stage and severity of the disease, the generalstate of health of the patient, and the judgment of the prescribingphysician.

The vaccine can be administered by methods well known in the art asdescribed in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997));Felgner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Felgner(U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S.Pat. No. 5,679,647, issued Oct. 21, 1997), the contents of all of whichare incorporated herein by reference in their entirety. The DNA of thevaccine can be complexed to particles or beads that can be administeredto an individual, for example, using a vaccine gun. One skilled in theart would know that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound, depends, for example,on the route of administration of the expression vector.

The vaccine can be delivered via a variety of routes. Typical deliveryroutes include parenteral administration, e.g., intradermal,intramuscular or subcutaneous delivery, optionally followed byelectroporation as described herein. Other routes include oraladministration, intranasal, and intravaginal routes. For the DNA of thevaccine in particular, the vaccine can be delivered to the interstitialspaces of tissues of an individual (Felgner et al., U.S. Pat. Nos.5,580,859 and 5,703,055, the contents of all of which are incorporatedherein by reference in their entirety). The vaccine can also beadministered to muscle, or can be administered via intradermal orsubcutaneous injections, or transdermally, such as by iontophoresis.Epidermal administration of the vaccine can also be employed. Epidermaladministration can involve mechanically or chemically irritating theoutermost layer of epidermis to stimulate an immune response to theirritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of whichare incorporated herein by reference in its entirety).

The vaccine can also be formulated for administration via the nasalpassages. Formulations suitable for nasal administration, wherein thecarrier is a solid, can include a coarse powder having a particle size,for example, in the range of about 10 to about 500 microns which isadministered in the manner in which snuff is taken, i.e., by rapidinhalation through the nasal passage from a container of the powder heldclose up to the nose. The formulation can be a nasal spray, nasal drops,or by aerosol administration by nebulizer. The formulation can includeaqueous or oily solutions of the vaccine.

The vaccine can be a liquid preparation such as a suspension, syrup orelixir. The vaccine can also be a preparation for parenteral,subcutaneous, intradermal, intramuscular or intravenous administration(e.g., injectable administration), such as a sterile suspension oremulsion.

The vaccine can be incorporated into liposomes, microspheres or otherpolymer matrices (Felgner et al., U.S. Pat. No. 5,703,055; Gregoriadis,Liposome Technology, Vols. I to III (2nd ed. 1993), the contents ofwhich are incorporated herein by reference in their entirety). Liposomescan consist of phospholipids or other lipids, and can be nontoxic,physiologically acceptable and metabolizable carriers that arerelatively simple to make and administer.

The vaccine can be administered via electroporation, such as by a methoddescribed in U.S. Pat. No. 7,664,545, the contents of which areincorporated herein by reference. The electroporation can be by a methodand/or apparatus described in U.S. Pat. Nos. 6,302,874; 5,676,646;6,241,701; 6,233,482; 6,216,034; 6,208,893; 6,192,270; 6,181,964;6,150,148; 6,120,493; 6,096,020; 6,068,650; and 5,702,359, the contentsof which are incorporated herein by reference in their entirety. Theelectroporation may be carried out via a minimally invasive device.

The minimally invasive electroporation device (“MID”) may be anapparatus for injecting the vaccine described above and associated fluidinto body tissue. The device may comprise a hollow needle, DNA cassette,and fluid delivery means, wherein the device is adapted to actuate thefluid delivery means in use so as to concurrently (for example,automatically) inject DNA into body tissue during insertion of theneedle into the said body tissue. This has the advantage that theability to inject the DNA and associated fluid gradually while theneedle is being inserted leads to a more even distribution of the fluidthrough the body tissue. The pain experienced during injection may bereduced due to the distribution of the DNA being injected over a largerarea.

The MID may inject the vaccine into tissue without the use of a needle.The MID may inject the vaccine as a small stream or jet with such forcethat the vaccine pierces the surface of the tissue and enters theunderlying tissue and/or muscle. The force behind the small stream orjet may be provided by expansion of a compressed gas, such as carbondioxide through a micro-orifice within a fraction of a second. Examplesof minimally invasive electroporation devices, and methods of usingthem, are described in published U.S. Patent Application No.20080234655; U.S. Pat. Nos. 6,520,950; 7,171,264; 6,208,893; 6,009,347;6,120,493; 7,245,963; 7,328,064; and 6,763,264, the contents of each ofwhich are herein incorporated by reference.

The MID may comprise an injector that creates a high-speed jet of liquidthat painlessly pierces the tissue. Such needle-free injectors arecommercially available. Examples of needle-free injectors that can beutilized herein include those described in U.S. Pat. Nos. 3,805,783;4,447,223; 5,505,697; and 4,342,310, the contents of each of which areherein incorporated by reference.

A desired vaccine in a form suitable for direct or indirectelectrotransport may be introduced (e.g., injected) using a needle-freeinjector into the tissue to be treated, usually by contacting the tissuesurface with the injector so as to actuate delivery of a jet of theagent, with sufficient force to cause penetration of the vaccine intothe tissue. For example, if the tissue to be treated is mucosa, skin ormuscle, the agent is projected towards the mucosal or skin surface withsufficient force to cause the agent to penetrate through the stratumcorneum and into dermal layers, or into underlying tissue and muscle,respectively.

Needle-free injectors are well suited to deliver vaccines to all typesof tissues, particularly to skin and mucosa. In some embodiments, aneedle-free injector may be used to propel a liquid that contains thevaccine to the surface and into the subject's skin or mucosa.Representative examples of the various types of tissues that can betreated using the invention methods include pancreas, larynx,nasopharynx, hypopharynx, oropharynx, lip, throat, lung, heart, kidney,muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue,ovary, blood vessels, or any combination thereof.

The MID may have needle electrodes that electroporate the tissue. Bypulsing between multiple pairs of electrodes in a multiple electrodearray, for example set up in rectangular or square patterns, providesimproved results over that of pulsing between a pair of electrodes.Disclosed, for example, in U.S. Pat. No. 5,702,359 entitled “NeedleElectrodes for Mediated Delivery of Drugs and Genes” is an array ofneedles wherein a plurality of pairs of needles may be pulsed during thetherapeutic treatment. In that application, which is incorporated hereinby reference as though fully set forth, needles were disposed in acircular array, but have connectors and switching apparatus enabling apulsing between opposing pairs of needle electrodes. A pair of needleelectrodes for delivering recombinant expression vectors to cells may beused. Such a device and system is described in U.S. Pat. No. 6,763,264,the contents of which are herein incorporated by reference.Alternatively, a single needle device may be used that allows injectionof the DNA and electroporation with a single needle resembling a normalinjection needle and applies pulses of lower voltage than thosedelivered by presently used devices, thus reducing the electricalsensation experienced by the patient.

The MID may comprise one or more electrode arrays. The arrays maycomprise two or more needles of the same diameter or differentdiameters. The needles may be evenly or unevenly spaced apart. Theneedles may be between 0.005 inches and 0.03 inches, between 0.01 inchesand 0.025 inches; or between 0.015 inches and 0.020 inches. The needlemay be 0.0175 inches in diameter. The needles may be 0.5 mm, 1.0 mm, 1.5mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more spaced apart.

The MID may consist of a pulse generator and a two or more-needlevaccine injectors that deliver the vaccine and electroporation pulses ina single step. The pulse generator may allow for flexible programming ofpulse and injection parameters via a flash card operated personalcomputer, as well as comprehensive recording and storage ofelectroporation and patient data. The pulse generator may deliver avariety of volt pulses during short periods of time. For example, thepulse generator may deliver three 15 volt pulses of 100 ms in duration.An example of such a MID is the Elgen 1000 system by Inovio BiomedicalCorporation, which is described in U.S. Pat. No. 7,328,064, the contentsof which are herein incorporated by reference.

The MID may be a CELLECTRA® (Inovio Pharmaceuticals, Blue Bell Pa.)device and system, which is a modular electrode system, that facilitatesthe introduction of a macromolecule, such as a DNA, into cells of aselected tissue in a body or plant. The modular electrode system maycomprise a plurality of needle electrodes; a hypodermic needle; anelectrical connector that provides a conductive link from a programmableconstant-current pulse controller to the plurality of needle electrodes;and a power source. An operator can grasp the plurality of needleelectrodes that are mounted on a support structure and firmly insertthem into the selected tissue in a body or plant. The macromolecules arethen delivered via the hypodermic needle into the selected tissue. Theprogrammable constant-current pulse controller is activated andconstant-current electrical pulse is applied to the plurality of needleelectrodes. The applied constant-current electrical pulse facilitatesthe introduction of the macromolecule into the cell between theplurality of electrodes. Cell death due to overheating of cells isminimized by limiting the power dissipation in the tissue by virtue ofconstant-current pulses. The Cellectra® device and system is describedin U.S. Pat. No. 7,245,963, the contents of which are hereinincorporated by reference. The CELLECTRA® device may be the CELLECTRA2000® device or CELLECTRA® 3PSP device.

The MID may be an Elgen 1000 system (Inovio Pharmaceuticals). The Elgen1000 system may comprise device that provides a hollow needle; and fluiddelivery means, wherein the apparatus is adapted to actuate the fluiddelivery means in use so as to concurrently (for example automatically)inject fluid, the described vaccine herein, into body tissue duringinsertion of the needle into the said body tissue. The advantage is theability to inject the fluid gradually while the needle is being insertedleads to a more even distribution of the fluid through the body tissue.It is also believed that the pain experienced during injection isreduced due to the distribution of the volume of fluid being injectedover a larger area.

In addition, the automatic injection of fluid facilitates automaticmonitoring and registration of an actual dose of fluid injected. Thisdata can be stored by a control unit for documentation purposes ifdesired.

It will be appreciated that the rate of injection could be either linearor non-linear and that the injection may be carried out after theneedles have been inserted through the skin of the subject to be treatedand while they are inserted further into the body tissue.

Suitable tissues into which fluid may be injected by the apparatus ofthe present invention include tumor tissue, skin or liver tissue but maybe muscle tissue.

The apparatus further comprises needle insertion means for guidinginsertion of the needle into the body tissue. The rate of fluidinjection is controlled by the rate of needle insertion. This has theadvantage that both the needle insertion and injection of fluid can becontrolled such that the rate of insertion can be matched to the rate ofinjection as desired. It also makes the apparatus easier for a user tooperate. If desired means for automatically inserting the needle intobody tissue could be provided.

A user could choose when to commence injection of fluid. Ideallyhowever, injection is commenced when the tip of the needle has reachedmuscle tissue and the apparatus may include means for sensing when theneedle has been inserted to a sufficient depth for injection of thefluid to commence. This means that injection of fluid can be prompted tocommence automatically when the needle has reached a desired depth(which will normally be the depth at which muscle tissue begins). Thedepth at which muscle tissue begins could for example be taken to be apreset needle insertion depth such as a value of 4 mm which would bedeemed sufficient for the needle to get through the skin layer.

The sensing means may comprise an ultrasound probe. The sensing meansmay comprise a means for sensing a change in impedance or resistance. Inthis case, the means may not as such record the depth of the needle inthe body tissue but will rather be adapted to sense a change inimpedance or resistance as the needle moves from a different type ofbody tissue into muscle. Either of these alternatives provides arelatively accurate and simple to operate means of sensing thatinjection may commence. The depth of insertion of the needle can furtherbe recorded if desired and could be used to control injection of fluidsuch that the volume of fluid to be injected is determined as the depthof needle insertion is being recorded.

The apparatus may further comprise: a base for supporting the needle;and a housing for receiving the base therein, wherein the base ismoveable relative to the housing such that the needle is retractedwithin the housing when the base is in a first rearward positionrelative to the housing and the needle extends out of the housing whenthe base is in a second forward position within the housing. This isadvantageous for a user as the housing can be lined up on the skin of apatient, and the needles can then be inserted into the patient's skin bymoving the housing relative to the base.

As stated above, it is desirable to achieve a controlled rate of fluidinjection such that the fluid is evenly distributed over the length ofthe needle as it is inserted into the skin. The fluid delivery means maycomprise piston driving means adapted to inject fluid at a controlledrate. The piston driving means could for example be activated by a servomotor. However, the piston driving means may be actuated by the basebeing moved in the axial direction relative to the housing. It will beappreciated that alternative means for fluid delivery could be provided.Thus, for example, a closed container which can be squeezed for fluiddelivery at a controlled or non-controlled rate could be provided in theplace of a syringe and piston system.

The apparatus described above could be used for any type of injection.It is however envisaged to be particularly useful in the field ofelectroporation and so it may further comprises means for applying avoltage to the needle. This allows the needle to be used not only forinjection but also as an electrode during, electroporation. This isparticularly advantageous as it means that the electric field is appliedto the same area as the injected fluid. There has traditionally been aproblem with electroporation in that it is very difficult to accuratelyalign an electrode with previously injected fluid and so users havetended to inject a larger volume of fluid than is required over a largerarea and to apply an electric field over a higher area to attempt toguarantee an overlap between the injected substance and the electricfield. Using the present invention, both the volume of fluid injectedand the size of electric field applied may be reduced while achieving agood fit between the electric field and the fluid.

Use in Combination

In some embodiments, the present invention provides a method of treatingSARS-CoV-2 infection, or treating, protecting against, and/or preventinga disease or disorder associated with SARS-CoV-2 infection in a subjectin need thereof by administering a combination of a nucleic acidmolecule encoding a SARS-CoV-2 antigen, or fragment or variant thereofin combination with one or more additional agents for the treatment ofSARS-CoV-2 infection or the treatment or prevention of disease ordisorder associated with SARS-CoV-2 infection. In some embodiments, thedisease or disorder associated with SARS-CoV-2 infection is CoronavirusDisease 2019 (COVID-19), Multisystem inflammatory syndrome in adults(MIS-A), or Multisystem inflammatory syndrome in children (MIS-C).

The nucleic acid molecule encoding a SARS-CoV-2 antigen and additionalagent may be administered using any suitable method such that acombination of the nucleic acid molecule encoding a SARS-CoV-2 antigenand the additional agent are both present in the subject. In oneembodiment, the method may comprise administration of a firstcomposition comprising an agent for the treatment of SARS-CoV-2infection or the treatment or prevention of disease or disorderassociated with SARS-CoV-2 infection and administration of a secondcomposition comprising a nucleic acid molecule encoding a SARS-CoV-2antigen less than 1, less than 2, less than 3, less than 4, less than 5,less than 6, less than 7, less than 8, less than 9 or less than 10 daysfollowing administration of the first composition comprising the agentfor the treatment of SARS-CoV-2 infection or the treatment or preventionof disease or disorder associated with SARS-CoV-2 infection. In oneembodiment, the method may comprise administration of a firstcomposition comprising a nucleic acid molecule encoding a SARS-CoV-2antigen and administration of a second composition comprising an agentfor the treatment of SARS-CoV-2 infection or the treatment or preventionof disease or disorder associated with SARS-CoV-2 infection less than 1,less than 2, less than 3, less than 4, less than 5, less than 6, lessthan 7, less than 8, less than 9 or less than 10 days followingadministration of the nucleic acid molecule encoding a SARS-CoV-2antigen. In one embodiment, the method may comprise administration of afirst composition comprising an agent for the treatment of SARS-CoV-2infection or the treatment or prevention of disease or disorderassociated with SARS-CoV-2 infection and a second composition comprisinga nucleic acid molecule encoding a SARS-CoV-2 antigen concurrently. Inone embodiment, the method may comprise administration of a singlecomposition comprising an agent for the treatment of SARS-CoV-2infection or the treatment or prevention of disease or disorderassociated with SARS-CoV-2 infection and a nucleic acid moleculeencoding a SARS-CoV-2 antigen.

In some embodiments, the agent for the treatment of SARS-CoV-2 infectionor the treatment or prevention of disease or disorder associated withSARS-CoV-2 infection is a therapeutic agent. In one embodiment, thetherapeutic agent is an antiviral agent. In one embodiment, thetherapeutic agent is an antibiotic agent.

Non-limiting examples of antibiotics that can be used in combinationwith the a nucleic acid molecule encoding a SARS-CoV-2 antigen of theinvention include aminoglycosides (e.g., gentamicin, amikacin,tobramycin), quinolones (e.g., ciprofloxacin, levofloxacin),cephalosporins (e.g., ceftazidime, cefepime, cefoperazone, cefpirome,ceftobiprole), antipseudomonal penicillins: carboxypenicillins (e.g.,carbenicillin and ticarcillin) and ureidopenicillins (e.g., mezlocillin,azlocillin, and piperacillin), carbapenems (e.g., meropenem, imipenem,doripenem), polymyxins (e.g., polymyxin B and colistin) and monobactams(e.g., aztreonam).

Administration as a Booster

In one embodiment, the immunogenic composition is administered as abooster vaccine following administration of an initial agent or vaccinefor the treatment of SARS-CoV-2 infection or the treatment or preventionof a disease or disorder associated with SARS-CoV-2 infection,including, but not limited to COVID-19, Multisystem inflammatorysyndrome in adults (MIS-A), or Multisystem inflammatory syndrome inchildren (MIS-C). In one embodiment, the booster vaccine is administeredat least once, at least twice, at least 3 times, at least 4 times, or atleast 5 times following administration of an initial agent or vaccinefor the treatment of SARS-CoV-2 infection or the treatment or preventionof a disease or disorder associated with SARS-CoV-2 infection,including, but not limited to COVID-19, Multisystem inflammatorysyndrome in adults (MIS-A), or Multisystem inflammatory syndrome inchildren (MIS-C). In one embodiment, the booster vaccine is administeredat least 8 hours, at least 12 hours, at least 16 hours, at least 20hours, at least 24 hours, at least 36 hours, at least 48 hours, at least60 hours, at least 72 hours, at least 4 days, at least 5 days, at least6 days, at least 1 week at least 2 weeks, at least 3 weeks, at least 4weeks, at least 1 month, at least 2 months, at least 3 months, at least4 months, at least 5 months, at least 6 months, at least 7 months, atleast 8 months, at least 9 months, at least 10 months, at least 11months, at least 1 year or greater than 1 year following administrationof an initial agent or vaccine for the treatment of SARS-CoV-2 infectionor the treatment or prevention of a disease or disorder associated withSARS-CoV-2 infection, including, but not limited to COVID-19,Multisystem inflammatory syndrome in adults (MIS-A), or Multisysteminflammatory syndrome in children (MIS-C).

Use in Assays

In some embodiments, the nucleic acid molecules, or encoded antigens, ofthe invention can be used in assays in vivo or in vitro. In someembodiments, the nucleic acid molecules, or encoded antigens can be usedin assays for detecting the presence of anti-SARS-CoV-2 spikeantibodies. Exemplary assays in which the nucleic acid molecules orencoded antigens can be incorporated into include, but are not limitedto, Western blot, dot blot, surface plasmon resonance methods, FlowCytometry methods, various immunoassays, for example,immunohistochemistry assays, immunocytochemistry assays, ELISA, captureELISA, enzyme-linked immunospot (ELISpot) assays, sandwich assays,enzyme immunoassay, radioimmunoassay, fluorescent immunoassay, and thelike, all of which are known to those of skill in the art. See e.g.Harlow et al., 1988, Antibodies: A Laboratory Manual, Cold SpringHarbor, New York; Harlow et al., 1999, Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY.

In one embodiment, the SARS-CoV-2 spike antigen, or fragments thereof,of the invention can be used in an assay for intracellular cytokinestaining combined with flow cytometry, to assess T-cell immuneresponses. This assay enables the simultaneous assessment of multiplephenotypic, differentiation and functional parameters pertaining toresponding T-cells, most notably, the expression of multiple effectorcytokines. These attributes make the technique particularly suitable forthe assessment of T-cell immune responses induced by the vaccine of theinvention.

In one embodiment, the SARS-CoV-2 spike antigen, or fragments thereof,of the invention can be used in an ELIspot assay. The ELISpot assay is ahighly sensitive immunoassay that measures the frequency ofcytokine-secreting cells at the single-cell level. In this assay, cellsare cultured on a surface coated with a specific capture antibody in thepresence or absence of stimuli. In one embodiment, the SARS-CoV-2 spikeantigen, or fragments thereof, of the invention can be used as thestimulus in the ELISpot assay.

Diagnostic Methods

In some embodiments, the invention relates to methods of diagnosing asubject as having SARS-CoV-2 infection or having SARS-CoV-2 antibodies.In some embodiments, the methods include contacting a sample from asubject with a SARS-CoV-2 antigen of the invention, or a cell comprisinga nucleic acid molecule for expression of the SARS-CoV-2 antigen, anddetecting binding of an anti-SARS-CoV-2 spike antibody to the SARS-CoV-2antigen of the invention. In such an embodiment, binding of ananti-SARS-CoV-2 spike antibody present in the sample of the subject tothe antigen, or fragment thereof, of the invention would indicate thatthe subject is currently infected or was previously infected withSARS-CoV-2.

Kits and Articles of Manufacture

Provided herein is a kit, which can be used for treating a subject usingthe method of vaccination described above. The kit can comprise theimmunogenic composition described herein.

The kit can also comprise instructions for carrying out the vaccinationmethod described above and/or how to use the kit. Instructions includedin the kit can be affixed to packaging material or can be included as apackage insert. While instructions are typically written or printedmaterials, they are not limited to such. Any medium capable of storinginstructions and communicating them to an end user is contemplated bythis disclosure. Such media include, but are not limited to, electronicstorage media (e.g., magnetic discs, tapes, cartridges), optical media(e.g., CD ROM), and the like. As used herein, the term “instructions”can include the address of an internet site which provides instructions.

Further provided herein are articles of manufacture containing theimmunogenic composition described herein. In some embodiments, thearticle of manufacture is a container, such as a vial, optionally asingle-use vial. In one embodiment, the article of manufacture is asingle-use glass vial equipped with a stopper, which contains theimmunogenic composition described herein to be administered. In someembodiments, the vial comprises a stopper, pierceable by a syringe, anda seal. In some embodiments, the article of manufacture is a syringe.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

EXAMPLES Example 1

Materials & Methods:

Cell lines. Human embryonic kidney (HEK)-293T (ATCC® CRL-3216™) andAfrican green monkey kidney COS-7 (ATCC® CRL-1651™) cell lines wereobtained from ATCC (Old Town Manassas, VA). All cell lines weremaintained in DMEM supplemented with 10% fetal bovine serum (FBS) andpenicillin-streptomycin.

In vitro protein expression (Western blot). Human embryonic kidneycells, 293T were cultured and transfected as described previously (Yan,et al. Enhanced cellular immune responses elicited by an engineeredHIV-1 subtype B consensus-based envelope DNA vaccine. Mol Ther. 2007;15(2):411-421.). 293T cells were transfected with pDNA usingTurboFectin8.0 (OriGene) transfection reagent following themanufacturer's protocol. Forty-eight hours later, cell lysates wereharvested using modified RIPA cell lysis buffer. Proteins were separatedon a 4-12% BIS-TRIS gel (ThermoFisher Scientific). Following transfer,blots were incubated with an anti-SARS-CoV spike protein polyclonalantibody (Novus Biologicals), and then visualized with horseradishperoxidase (HRP)-conjugated anti-mouse IgG (GE Amersham).

Immunofluorescence of transfected 293T cells. For in vitro staining ofSpike protein expression, 293T cells were cultured on 4-well glassslides (Lab-Tek) and transfected with 3 μg/well of pDNA usingTurboFectin8.0 (OriGene) transfection reagent following themanufacturer's protocol. Cells were fixed 48 hrs after transfection with10% Neutral-buffered Formalin (BBC Biochemical, Washington State) for 10min at room temperature (RT) and then washed with PBS. Before staining,chamber slides were blocked with 0.3% (v/v) Triton-X (Sigma), 2% (v/v)donkey serum in PBS for 1 hr at RT. Cells were stained with a rabbitanti-SARS-CoV spike protein polyclonal antibody (Novus Biologicals)diluted in 1% (w/v) BSA (Sigma), 2% (v/v) donkey serum, 0.3% (v/v)Triton-X (Sigma) and 0.025% (v/v) 1 g/ml Sodium Azide (Sigma) in PBS for2 hrs at RT. Slides were washed three times for 5 min in PBS and thenstained with donkey anti-rabbit IgG AF488 (Life Technologies, A21206)for 1 hr at RT. Slides were washed again and mounted and covered withDAPI-Fluoromount (SouthernBiotech).

In vitro RNA expression (qRT-PCR). In vitro mRNA expression of theplasmid was demonstrated by transfection of COS-7 with serially dilutedplasmids followed by analysis of the total RNA extracted from the cellsusing reverse transcription and PCR. Transfections of fourconcentrations of the plasmid were performed using FuGENE® 6transfection reagent (Promega) which resulted in final masses rangingbetween 80 and 10 ng/well. The transfections were performed induplicate. Following 18 to 26 hours of incubation, the cells were lysedwith RLT Buffer (Qiagen). Total RNA was isolated from each well usingthe Qiagen RNeasy kit following the kit instructions. The resulting RNAconcentration was determined by OD_(260/280), and samples of the RNAwere diluted to 10 ng/4. One hundred nanograms of RNA was then convertedto cDNA using the High Capacity cDNA Reverse Transcription (RT) kit(Applied Biosystems) following the kit instructions. RT reactionscontaining RNA but no reverse transcriptase (minus RT) were included ascontrols for plasmid DNA or cellular genomic DNA sample contamination.Eight μL of sample cDNA were then subjected to PCR using primers andprobes that are specific to the target sequence (pGX9501Forward—CAGGACAAGAACACACAGGAA (SEQ ID NO: 7); pGX9501Reverse—CAGGCAGGATTTGGGAGAAA (SEQ ID NO: 8); pGX9501Probe—ACCCATCAAGGACTTTGGAGG (SEQ ID NO: 9); and pGX9503Forward—AGGACAAGAACACACAGGAAG (SEQ ID NO: 10); pGX9503Reverse—CAGGATCTGGGAGAAGTTGAAG (SEQ ID NO: 11); pGX9503Probe—ACACCACCCATCAAGGACTTTGGA (SEQ ID NO: 12)). In a separate reaction,the same quantity of sample cDNA was subjected to PCR using primers anda probe designed for COS-7 cell line β-actin sequences (β3-actinForward—GTGACGTGGACATCCGTAAA (SEQ ID NO: 13); β-actinReverse—CAGGGCAGTAATCTCCTTCTG (SEQ ID NO: 14); β-actinProbe—TACCCTGGCATTGCTGACAGGATG (SEQ ID NO: 15)). The primers and probeswere synthesized by Integrated DNA Technologies, Inc. and the probeswere labeled with 56-FAM and Black Hole Quencher 1. The reaction usedABI Fast Advance 2X (Cat. No. 4444557), with final forward and reverseprimer concentrations of 1 μM and probe concentrations of 0.3 μM. Usinga QuantStudio™ 7 Flex Real Time PCR Studio System (Applied Biosystems),samples were first subjected to a hold of 1 minute at 95° C. and then 40cycles of PCR with each cycle consisting of 1 second at 95° C. and 20seconds at 60° C. Following PCR, the amplifications results wereanalyzed as follows. The negative transfection controls (NTCs), theminus RT controls, and the NTC were scrutinized for each of theirrespective indications. The threshold cycle (C_(T)) of each transfectionconcentration for the INO-4800 SARS-CoV-2 target mRNA and for theβ-actin mRNA was generated from the QuantStudio™ software using anautomatic threshold setting. The plasmid was considered to be active formRNA expression if the expression in any of the plasmid-transfectedwells compared to the negative transfection controls were greater than 5C_(T). Animals. Female, 6 week old C57/BL6 and BALB/c mice werepurchased from Charles River Laboratories (Malvern, PA) and The JacksonLaboratory (Bar Harbor, ME). Female, 8 week old Hartley guinea pigs werepurchased from Elm Hill Labs (Chelmsford, MA). All animals were housedin the animal facility at The Wistar Institute Animal Facility orAcculab Life Sciences (San Diego, CA). All animal testing and researchcomplied with all relevant ethical regulations and studies receivedethical approval by the Wistar Institute or Acculab Institutional AnimalCare and Use Committees (IACUC). For mouse studies, on day 0, doses of2.5, 10 or 25 μg pDNA were administered to the tibialis anterior (TA)muscle by needle injection followed by CELLECTRA® in vivoelectroporation (EP). The CELLECTRA® EP delivery consists of two sets ofpulses with 0.2 Amp constant current. Second pulse sets is delayed 3seconds. Within each set there are two 52 ms pulses with a 198 ms delaybetween the pulses. On days 0 and 14, blood was collected. Parallelgroups of mice were serially sacrificed on days 4, 7, and 10post-immunization for analysis of cellular immune responses. For guineapig studies, on day 0, 100 μg pDNA was administered to the skin byMantoux injection followed by CELLECTRA® in vivo EP.

Antigen binding ELISA. ELISAs were performed to determine sera antibodybinding titers. Nunc ELISA plates were coated with 1 μg/ml recombinantprotein antigens in Dulbecco's phosphate-buffered saline (DPBS)overnight at 4° C. Plates were washed three times, then blocked with 3%bovine serum albumin (BSA) in DPBS with 0.05% Tween 20 for 2 hours at37° C. Plates were then washed and incubated with serial dilutions ofmouse or guinea pig sera and incubated for 2 hours at 37° C. Plates wereagain washed and then incubated with 1:10,000 dilution of horse radishperoxidase (HRP)-conjugated anti-guinea pig IgG secondary antibody(Sigma-Aldrich, cat. A7289) or HRP-conjugated anti-mouse IgG secondaryantibody (Sigma-Aldrich) and incubated for 1 hour at RT. After finalwash, plates were developed using SureBlue™ TMB 1-Component PeroxidaseSubstrate (KPL, cat. 52-00-03), and the reaction stopped with TMB StopSolution (KPL, cat. 50-85-06). Plates were read at 450 nm wavelengthwithin 30 minutes using a Synergy™ HTX plate reader (BioTek Instruments,Highland Park, VT). Binding antibody endpoint titers (EPTs) werecalculated as previously described (Bagarazzi M L, Yan J, Morrow M P, etal. Immunotherapy against HPV16/18 generates potent TH1 and cytotoxiccellular immune responses. Sci Transl Med. 2012; 4(155):155ra138).Binding antigens tested included, SARS-CoV-2 antigens: 51 spike protein(Sino Biological 40591-VO8H), S1+S2 ECD spike protein (Sino Biological40589-VO8B1), RBD (University of Texas, at Austin (McLellan Lab.));SARS-COV antigens: Spike S1 protein (Sino Biological 40150-VO8B1), S(1-1190) (Immune Tech IT-002-001P) and Spike C-terminal (Meridian LifeScience R18572).

ACE2 Competition ELISA. For mouse studies, ELISAs were performed todetermine sera IgG antibody competition against human ACE2 with a humanFc tag. Nunc ELISA plates were coated with 1 μg/mL rabbit anti-His6× in1×PBS for 4-6 hours at room temperature (RT) and washed 4 times withwashing buffer (1×PBS and 0.05% Tween® 20). Plates were blockedovernight at 4° C. with blocking buffer (1×PBS, 0.05% Tween® 20, 5%evaporated milk and 1% FBS). Plates were washed four times with washingbuffer then incubated with full length (S1+S2) spike protein containinga C-terminal His tag (Sino Biologics, cat. 40589-V08B1) at 10 μg mL-1for 1 hour at RT. Plates were washed and then serial dilutions ofpurified mouse IgG mixed with 0.1 μg mL-1 recombinant human ACE2 with ahuman Fc tag (ACE2-IgHu) were incubated for 1-2 hours at RT. Plates wereagain washed and then incubated with 1:10,000 dilution of horse radishperoxidase (HRP) conjugated anti-human IgG secondary antibody (Bethyl,cat. A80-304P) and incubated for 1 hour at RT. After final wash plateswere developed using 1-Step Ultra TMB-ELISA Substrate (Thermo, cat.34029) and the reaction stopped with 1 M Sulfuric Acid. Plates were readat 450 nm wavelength within 30 minutes using a SpectraMax Plus 384Microplate Reader (Molecular Devices, Sunnyvale, CA). Competition curveswere plotted and the area under the curve (AUC) was calculated usingPrism 8 analysis software with multiple t-tests to determine statisticalsignificance.

For guinea pig studies, 96 well half area assay plates (Costar) werecoated with 25 μl per well of 5 μg/mL of SARS-CoV-2 spike S1+S2 protein(Sino Biological) diluted in 1×DPBS (Thermofisher) overnight at 4° C.Plates were washed with 1×PBS buffer with 0.05% TWEEN® 20 (Sigma). 100μl per well of 3% (w/v) BSA (Sigma) in 1×PBS with 0.05% TWEEN® 20 wereadded and incubated for 1 hr at 37° C. Serum samples were diluted 1:20in 1% (w/v) BSA in 1×PBS with 0.05% TWEEN. After washing the assayplate, 25 μl/well of diluted serum was added and incubated 1 hr at 37°C. Human recombinant ACE2-Fc-tag (Sinobiological) was added directly tothe diluted serum, followed by 1 hr of incubation at 37° C. Plates werewashed and 25 μl per well of 1:10,000 diluted goat anti-hu Fc fragmentantibody HRP (Bethyl, A80-304P) was added to the assay plate. Plateswere incubated 1 hr at RT. For development the SureBlue/TMB StopSolution (KPL, MD) was used and O.D. was recorded at 450 nm.

SARS-CoV-2 Pseudovirus neutralization assay. SARS-CoV-2 pseudotypedviruses were produced using HEK293T cells transfected with GeneJammer(Agilent) using IgE-SARS-CoV-2 S plasmid (Genscript) and pNL4-3.Luc.R-E-plasmid (NIH AIDS reagent) at a 1:1 ratio. Forty-eight hours posttransfection, transfection supernatant was collected, enriched with FBSto 12% final volume, steri-filtered (Millipore Sigma), and aliquoted forstorage at −80° C. SARS-CoV-2 pseudotyped viruses were titered andyielded greater than 50 times the relative luminescence units (RLU) tocells alone after 72h of infection. Mouse sera from INO-4800 vaccinatedand naive groups were heat inactivated for 15 minutes at 56° C. andserially diluted three fold starting at a 1:10 dilution for assay. Serawere incubated with a fixed amount of SARS-CoV-2 pseudotyped virus for90 minutes. HEK293T cells stably expressing ACE2 were added after 90minutes and allowed to incubate in standard incubator (37% humidity, 5%CO₂) for 72 hours. Post infection, cells were lysed using Britelite™plus luminescence reporter gene assay system (Perkin Elmer Catalog no.6066769) and relative luminescence units (RLU) were measured using theBiotek plate reader. Neutralization titers (ID₅₀) were calculated as theserum dilution at which RLU were reduced by 50% compared to RLU in viruscontrol wells after subtraction of background RLU in cell control wells.

SARS-CoV-2 wildtype virus neutralization assays.SARS-CoV-2/Australia/VIC01/2020 isolate neutralization assays wereperformed at Public Health England (Porton Down, UK). Neutralizing virustiters were measured in serum samples that had been heat-inactivated at56° C. for 30 minutes. SARS-CoV-2 (Australia/VIC01/2020 isolate) (Calyet al., Isolation and rapid sharing of the 2019 novel coronavirus(SARS-CoV-2) from the first patient diagnosed with COVID-19 inAustralia. Med. J. Aust. (2020) doi: 10.5694/mja2.50569; Publishedonline: 13 Apr. 2020) was diluted to a concentration of 933 pfu/ml andmixed 50:50 in 1% FCS/MEM containing 25 mM HEPES buffer with doublingserum dilutions from 1:10 to 1:320 in a 96-well V-bottomed plate. Theplate was incubated at 37° C. in a humidified box for 1 hour before thevirus was transferred into the wells of a twice DPBS-washed 24-wellplate that had been seeded the previous day at 1.5×10⁵ Vero E6 cells perwell in 10% FCS/MEM. Virus was allowed to adsorb at 37° C. for a furtherhour and overlaid with plaque assay overlay media (1×MEM/1.5% CMC/4% FCSfinal). After 5 days incubation at 37° C. in a humidified box, theplates were fixed, stained and plaques counted. Median neutralizingtiters (ND50) were determined using the Spearman-Karber formula relativeto virus only control wells.

SARS-CoV-2/WH-09/human/2020 isolate neutralization assays were performedat the Institute of Laboratory Animal Science, Chinese Academy ofMedical Sciences (CAMS) approved by the National Health Commission ofthe People's Republic of China. Seed SARS-CoV-2(SARS-CoV-2/WH-09/human/2020) stocks and virus isolation studies wereperformed in Vero E6 cells, which are maintained in Dulbecco's modifiedEagle's medium (DMEM, Invitrogen, Carlsbad, USA) supplemented with 10%fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/mlstreptomycin, and incubated at 36.5° C., 5% CO₂. Virus titer weredetermined using a standard 50% tissue culture infection dose (TCID50)assay. Serum samples collected from immunized animals were inactivatedat 56° C. for 30 minutes and serially diluted with cell culture mediumin two-fold steps. The diluted samples were mixed with a virussuspension of 100 TCID50 in 96-well plates at a ratio of 1:1, followedby 2 hours incubation at 36.5° C. in a 5% CO₂ incubator. 1-2×10⁴ Verocells were then added to the serum-virus mixture, and the plates wereincubated for 3-5 days at 36.5° C. in a 5% CO₂ incubator. Cytopathiceffect (CPE) of each well was recorded under microscopes, and theneutralizing titer was calculated by the dilution number of 50%protective condition.

Bronchoalveolar lavage collection. Bronchoalveolar lavage (BAL) fluidwas collected by washing the lungs of euthanized and exsanguinated micewith 700-1000 ul of ice-cold PBS containing 100 μm EDTA, 0.05% sodiumazide, 0.05% Tween® 20, and 1× protease inhibitor (Pierce) (mucosal prepsolutions (MPS)) with a blunt-ended needle. Guinea pig lungs were washedwith 20 ml of MPS via 16G catheter inserted into the trachea. CollectedBAL fluid was stored at −20° C. until the time of assay.

IFN-γ ELISpot. Mice: Spleens from mice were collected individually inRPMI1640 media supplemented with 10% FBS (R10) andpenicillin/streptomycin and processed into single cell suspensions. Cellpellets were re-suspended in 5 mL of ACK lysis buffer (LifeTechnologies, Carlsbad, CA) for 5 min at room temperature, and PBS wasthen added to stop the reaction. The samples were again centrifuged at1,500 g for 10 min, cell pellets re-suspended in R10, and then passedthrough a 45 μm nylon filter before use in ELISpot assay. ELISpot assayswere performed using the Mouse IFN-γ ELISpot^(PLUS) plates (MABTECH).96-well ELISpot plates pre-coated with capture antibody were blockedwith R10 medium overnight at 4° C. 200,000 mouse splenocytes were platedinto each well and stimulated for 20 hours with pools of 15-mer peptidesoverlapping by 9 amino acid from the SARS-CoV-2, SARS-CoV, or MERS-CoVSpike proteins (5 peptide pools per protein). Additionally, matrixmapping was performed using peptide pools in a matrix designed toidentify immunodominant responses. Cells were stimulated with a finalconcentration of 5 μL of each peptide/well in RPMI+10% FBS (R10). Thespots were developed based on manufacturer's instructions. R10 and cellstimulation cocktails (Invitrogen) were used for negative and positivecontrols, respectively. Spots were scanned and quantified by ImmunoSpot™CTL reader. Spot-forming unit (SFU) per million cells was calculated bysubtracting the negative control wells.

Flow cytometry. Intracellular cytokine staining was performed onsplenocytes harvested from BALB/c and C57BL/6 mice stimulated with theoverlapping peptides spanning the SARS-CoV-2 S protein for 6 hours at37° C., 5% CO₂. Cells were stained with the following antibodies from BDBiosciences, unless stated, with the dilutions stated in parentheses:FITC anti-mouse CD107a (1:100), PerCP-Cy5.5 anti-mouse CD4 (1:100), APCanti-mouse CD8a (1:100), ViViD Dye (1-40) (LIVE/DEAD® Fixable VioletDead Cell Stain kit; Invitrogen, L34955), APC-Cy7 anti-mouse CD3e(1:100), and BV605 anti-mouse IFN-γ (1:75) (eBiosciences). PhorbolMyristate Acetate (PMA) were used as a positive control, and completemedium only as the negative control. Cells were washed, fixed and, cellevents were acquired using an FACS CANTO (BD Biosciences), followed byFlowJo software (FlowJo LLC, Ashland, OR) analysis.

Statistics. All statistical analyses were performed using GraphPad Prism7 or 8 software (La Jolla, CA). These data were considered significantif p<0.05. The lines in all graphs represent the mean value and errorbars represent the standard deviation. No samples or animals wereexcluded from the analysis. Randomization was not performed for theanimal studies. Samples and animals were not blinded before performingeach experiment.

Results

Design and Synthesis of SARS-CoV-2 DNA Vaccine Constructs

Four spike protein sequences were retrieved from the first fouravailable SARS-CoV-2 full genome sequences published on GISAID (GlobalInitiative on Sharing All Influenza Data). Three Spike sequences were100% matched and one was considered an outlier (98.6% sequence identitywith the other sequences). After performing a sequence alignment, theSARS-CoV-2 spike glycoprotein sequence (“Covid-19 spike antigen”; SEQ IDNO: 1) was generated and an N-terminal IgE leader sequence was added.The highly optimized DNA sequence encoding SARS-CoV-2 IgE-spike wascreated as described elsewhere herein to enhance expression andimmunogenicity. SARS-CoV-2 spike outlier glycoprotein sequence(“Covid-19 spike-OL antigen”; SEQ ID NO: 4) was generated and anN-terminal IgE leader sequence was added. The optimized DNA sequence wassynthesized, digested with BamHI and XhoI, and cloned into theexpression vector pGX0001 under the control of the human cytomegalovirusimmediate-early promoter and a bovine growth hormone polyadenylationsignal. The resulting plasmids were designated as pGX9501 and pGX9503,designed to encode the SARS-CoV-2 S protein from the 3 matched sequencesand the outlier sequence, respectively (FIG. 1A).

In Vitro Characterization of Synthetic DNA Vaccine Constructs

Expression of the encoded SARS-CoV-2 spike transgene at the RNA level inCOS-7 cells transfected with pGX9501 and pGX9503 was measured. Using thetotal RNA extracted from the transfected COS-7 cells, expression of thespike transgene was confirmed by RT-PCR (FIG. 1B). In vitro spikeprotein expression in 293T cells was measured by Western blot analysisusing a cross-reactive antibody against SARS-CoV S protein on celllysates. Western blots of the lysates of HEK-293T cells transfected withpGX9501 or pGX9503 constructs revealed bands approximate to thepredicted S protein molecular weight, 140-142 kDa, with slight shiftslikely due to the 22 potential N-linked glycans in the S protein (FIG.1C). In immunofluorescent studies, the S protein was detected in 293Tcells transfected with pGX9501 or pGX9503 (FIG. 1D). In summary, invitro studies revealed the expression of the Spike protein at both theRNA and protein level after transfection of cell lines with thecandidate vaccine constructs.

Humoral immune responses in mice. pGX9501 was selected as the vaccineconstruct to advance to immunogenicity studies, due to the broadercoverage it would likely provide compared to the outlier, pGX9503.pGX9501 was subsequently termed INO-4800. The immunogenicity of INO-4800was evaluated in BALB/c mice, post-administration to the tibialisanterior muscle using the CELLECTRA® delivery device. (Sardesai &Weiner, Curr. Opin. Immunol., 23, 421-429 (2011). The reactivity of thesera from a group of mice immunized with INO-4800 was measured against apanel of SARS-CoV-2 and SARS-CoV antigens (FIG. 2 ). Analysis revealedIgG binding against SARS-CoV-2 S protein antigens, with limitedcross-reactivity to SARS-CoV S protein antigens in the sera ofINO-4800-immunized mice. The serum IgG binding endpoint titers in miceimmunized with pDNA against recombinant SARS-CoV-2 spike protein S1+S2regions (FIGS. 3A and 3B) and recombinant SARS-CoV-2 spike proteinreceptor binding domain (RBD) (FIGS. 3C and 3D) were measured. Endpointtiters were observed in the sera of mice at day 14 after immunizationwith a single dose of INO-4800 (FIGS. 3B, 3C, 3D).

Neutralization assay. A neutralization assay with a pNL4-3.Luc.R-E-basedpseudovirus displaying the SARS-CoV-2 Spike protein was developed.Neutralization titers were detected by a reduction in relativeluciferase units (RLU) compared to controls which had no decrease in RLUsignal. BALB/c mice were immunized twice with INO-4800, on days 0 and14, and sera was collected on day 7 post-2nd immunization. Thepseudovirus was incubated with serial dilutions of mouse sera and thesera-virus mixture was added to 293T cells stably expressing the humanACE2 receptor (ACE2-293T) for 72 hours. Neutralization ID50 averagetiters of 92.2 were observed in INO-4800 immunized mice (FIGS. 4A and4B). No reduction in RLU was observed for the control animals.Neutralizing titers were additionally measured against two wildtypeSARS-CoV-2 virus strains by plaque reduction neutralization test (PRNT)assay. Sera from INO-4800 immunized BALB/c mice neutralized bothSARS-CoV-2/WH-09/human/2020 and SARS-CoV-2/Australia/VIC01/2020 virusstrains with average ND50 titers of 97.5 and 128.1, respectively (Table1). Live virus neutralizing titers were also evaluated in C57BL/6 micefollowing the same INO-4800 immunization regimen. Sera from INO-4800immunized C57BL/6 mice neutralized wildtype SARS-CoV-2 virus withaverage ND50 titer of 340 (Table 1).

TABLE 1 Sera neutralizing activity after INO-4800 administration to miceand guinea pigs. Serum Sample ND50 Immunization Time Neutralization(Reciprocal Model Vaccine N Regimen point Assay Dilution) BALB/c pVAX 425 μg Day 21 SARS-CoV-2 <20, <20, Mouse Days 0, 14 (WH-09/human/2020)<20, <20 INO-4800 4 25 μg Day 21 SARS-CoV-2 30, 40, 80, Days 0, 14(WH-09/human/2020) 240 pVAX 8 25 μg Day 21 SARS-CoV-2 <10, 12, 13, 15,Days 0, 14 (Australia/VIC01/2020) 16, 17, 19, 24 INO-4800 8 25 μg Day 21SARS-CoV-2 27, 46, 91, 108, Days 0, 14 (Australia/VIC01/2020) 130, 161,221, 241 pVAX 5 10 μg Day 21 SARS-CoV-2 8, 8, 8, 8, 8 Days 0, 14Pseudovirus INO-4800 5 10 μg Day 21 SARS-CoV-2 43, 55, 87, Days 0, 14Pseudovirus 129, 147 C57BL/6 pVAX 4 25 μg Day 21 SARS-CoV-2 <20, <20,Mouse Days 0, 14 (WH-09/human/2020) <20, <20 INO-4800 4 25 μg Day 21SARS-CoV-2 240, 240, Days 0, 14 (WH-09/human/2020) 240, 640 Guinea pVAX5 100 μg Day 42 SARS-CoV-2 <10, 14, 20, Pig Days 0, 14, 28(Australia/VIC01/2020) 21, 25 INO-4800 5 100 μg Day 42SARS-CoV-2 >320, >320, >320, Days 0, 14, 28(Australia/VIC01/2020) >320, >320 pVAX 5 100 μg Day 35 SARS-CoV-2 <20,<20, <20, Days 0, 14, 28 Pseudovirus <20, <20 INO-4800 5 100 μg Day 35SARS-CoV-2 527, 532, 579, Days 0, 14, 28 Pseudovirus 614, 616 New SSC 5Days 0, 28 Day 42 SARS-CoV-2 <10, <10, <10, Zealand Pseudovirus <10, <10White INO-4800 5 1 mg, Days 0, Day 42 SARS-CoV-2 12, 23, 32, Rabbit 28Pseudovirus 148, 178 INO-4800 5 2 mg, Days 0. Day 42 SARS-CoV-2 202,237, 252, 28 Pseudovirus 455, 995 Non- INO-4800 5 1 mg, Days 0, Day 42SARS-CoV-2 15, 27, 55, human 28 Pseudovirus 61, 1489 primates INO-4800 52 mg, Days 0. Day 42 SARS-CoV-2 78, 23, 13, 28 Pseudovirus 48, <10

The immunogenicity of INO-4800 in the Hartley guinea pig model, anestablished model for intradermal vaccine delivery (Carter, et al. Theadjuvant GLA-AF enhances human intradermal vaccine responses. Sci Adv.2018; 4(9):eaas9930; Schultheis, et al. Characterization of guinea pig Tcell responses elicited after EP-assisted delivery of DNA vaccines tothe skin. Vaccine. 2017; 35(1):61-70), was assessed. 100 μg of pDNA wasadministered by Mantoux injection to the skin and followed by CELLECTRA®device on day as described in the methods section above. On day 14,anti-spike protein binding of serum antibodies was measured by ELISA.Immunization with INO-4800 revealed an immune response in respect toSARS-CoV-2 S1+2 protein binding IgG levels in the sera (FIGS. 5A and5B). The endpoint SARS-CoV-2 S protein binding titer at day 14 was10,530 and 21 in guinea pigs treated with 100 μg INO-4800 or pVAX(control), respectively (FIG. 5B). Antibody neutralizing activityfollowing intradermal INO-4800 immunization in the guinea pig model wasevaluated. Guinea pigs were treated on days 0, 14, and 28 with pVAX orINO-4800, and sera samples were collected on days 35 or 42 to measuresera neutralizing activity against pseudovirus or wildtype virus,respectively. SARS-CoV-2 pseudovirus neutralizing activity with averageND50 titers of 573.5 was observed for the INO-4800 immunized guinea pigs(Table 1). Wildtype SARS-CoV-2 virus activity was also observed for theINO-4800 immunized guinea pigs with ND50 titers >320 by PRNT assayobserved in all animals (Table 1). The functionality of the serumantibodies was further measured by assessing their ability to inhibitACE2 binding to SARS-CoV-2 spike protein. Serum (1:20 dilution)collected from INO-4800 immunized guinea pigs after 2nd immunizationinhibited binding of SARS-CoV-2 Spike protein over range ofconcentrations of ACE-2 (0.25 μg/ml through 4 μg/ml) (FIG. 6E).Furthermore, serum dilution curves revealed serum collected fromINO-4800 immunized guinea pigs blocked binding of ACE-2 to SARS-CoV-2 ina dilution-dependent manner (FIG. 6F). Serum collected from pVAX-treatedanimals displayed negligible activity in the inhibition of ACE-2 bindingto the virus protein, the decrease in OD signal at the highestconcentration of serum is considered a matrix effect in the assay.

Inhibition of SARS-CoV-2 S protein binding to ACE2 receptor. Thereceptor inhibiting functionality of INO-4800-induced antibody responseswas examined. An ELISA-based ACE2 inhibition assay was developed as asurrogate for neutralization. As a control in the assay, ACE2 is shownto bind to SARS-CoV-2 Spike protein with an EC50 of 0.025 μg/ml (FIG.6A). BALB/c mice were immunized on Days 0 and Day 14 with 10 μg ofINO-4800, and serum IgG was purified on Day 21 post-immunization toensure inhibition is antibody-mediated. Inhibition of the Spike-ACE2interaction using serum IgG from a naïve mouse and from an INO-4800vaccinated mouse were compared (FIG. 6B). The receptor inhibition assaywas repeated with a group of five immunized mice, demonstrating thatINO-4800-induced antibodies competed with ACE2 binding to the SARS-CoV-2Spike protein (FIGS. 6C and 6F). ACE2 binding inhibition was furtherevaluated in the guinea pig model. Sera collected from INO-4800immunized guinea pigs inhibited binding of SARS-CoV-2 Spike protein overrange of concentrations of ACE2 (0.25 μg/ml through 4 μg/ml) (FIG. 6D).Furthermore, serum dilution curves revealed sera collected from INO-4800immunized guinea pigs blocked binding of ACE2 to SARS-CoV-2 in adilution-dependent manner (FIG. 6E). Sera collected from pVAX-treatedanimals displayed negligible activity in the inhibition of ACE2 bindingto the virus protein, the decrease in OD signal at the highestconcentration of serum is considered a matrix effect in the assay. FIG.6F depicts IgGs purified from n=5 mice day 14 post second immunizationwith INO-4800 show competition against ACE2 receptor binding toSARS-CoV-2 Spike protein compared to pooled naïve mice IgGs.

In summary, immunogenicity testing in both mice and guinea pigs revealedthe SARS-CoV-2 vaccine candidate, INO-4800, was capable of elicitingantibody responses to SARS-CoV-2 spike protein. ACE2 is considered to bethe primary receptor for SARS-CoV-2 cellular entry, blocking thisinteraction suggests INO-4800-induced antibodies may prevent hostinfection.

Biodistribution of SARS-CoV-2 reactive IgG to the lung. Lowerrespiratory disease (LRD) is associated with severe cases of COVID-19.The presence of antibodies at the lung mucosa targeting SARS-CoV-2 couldpotentially mediate protection against LRD. The presence of SARS-CoV-2specific antibody in the lungs of immunized mice and guinea pigs wasevaluated. BALB/c mice and Hartley guinea pigs were immunized, on days 0and 14 or 0, 14 and 28, respectively, with INO-4800 or pVAX controlpDNA. Bronchoalveolar lavage (BAL) fluid was collected followingsacrifice, and SARS-CoV-2 S protein ELISAs were performed. In bothBALB/c and Hartley guinea pigs which received INO-4800, a statisticallysignificant increase in SARS-CoV-2 S protein binding IgG in BAL fluidcompared to animals receiving pVAX control was measured (FIGS. 7A-7D).Taken together, these data demonstrate the presence of anti-SARS-CoV-2specific antibody in the lungs following immunization with INO-4800.

Coronavirus cross-reactive cellular immune responses in mice. T cellresponses against SARS-CoV-2, SARS-CoV, and MERS-CoV S antigens wereassayed by IFN-γ ELISpot. Groups of BALB/c mice were sacrificed at days4, 7, or 10 post-INO-4800 administration (2.5 or 10 μg of pDNA),splenocytes were harvested, and a single-cell suspension was stimulatedfor 20 hours with pools of 15-mer overlapping peptides spanning theSARS-CoV-2, SARS-CoV, and MERS-CoV spike protein. Day 7 post-INO-4800administration, T cell responses of 205 and 552 SFU per 106 splenocytesagainst SARS-CoV-2 were measured for the 2.5 and 10 μg doses,respectively (FIG. 8A). Higher magnitude responses of 852 and 2,193 SFUper 106 splenocytes against SARS-CoV-2 were observed on Day 10post-INO-4800 administration. Additionally, the cross-reactivity of thecellular response elicited by INO-4800 against SARS-CoV was assayed,showing detectable, albeit lower, T cell responses on both Day 7 (74[2.5 μg dose] and 140 [10 μg dose] SFU per 106 splenocytes) and Day 10post-administration (242 [2.5 μg dose] and 588 [10 μg dose] SFU per 106splenocytes) (FIG. 8B). Interestingly, no cross-reactive T cellresponses were observed against MERS-CoV peptides (FIG. 8C).Representative images of the IFN-γ ELISpot plates are provided in FIG.31 . The T cell populations which were producing IFN-γ were identified.Flow cytometric analysis on splenocytes harvested from BALB/c mice onDay 14 after a single INO-4800 immunization revealed the T cellcompartment to contain 0.04% CD4+ and 0.32% CD8+IFN-γ+ T cells afterstimulation with SARS-CoV-2 antigens (FIG. 32 ).

BALB/c SARS-CoV-2 epitope mapping. Epitope mapping was performed on thesplenocytes from BALB/c mice receiving the 10 μg INO-4800 dose. Thirtymatrix mapping pools were used to stimulate splenocytes for 20 hours andimmunodominant responses were detected in multiple peptide pools (FIG.14A). The responses were deconvoluted to identify several epitopes(H2-Kd) clustering in the receptor binding domain and in the S2 domain(FIG. 14B). Interestingly, one SARS-CoV-2 H2-Kd epitope, PHGVVFLHV (SEQID NO: 16), was observed to be overlapping and adjacent to the SARS-CoVhuman HLA-A2 restricted epitope VVFLHVTVYV (SEQ ID NO: 17).

In summary, T cell responses against SARS-CoV-2 S protein epitopes weredetected in mice immunized with INO-4800.

Example 2—Cellular and Humoral Immune Responses Measured inINO-4800-Treated New Zealand White (NZW) Rabbits

Day 0 and 28 intradermal delivery of pDNA. PBMC IFN-γ ELISpot (FIG. 9 );Serum IgG binding ELISA (FIG. 10 ).

Example 3 Humoral Immune Responses to SARS-CoV-2 Spike Protein Measuredin INO-4800 Treated in Rhesus Monkeys

Day 0 and 28 intradermal delivery of pDNA. Serum IgG binding ELISA.(FIGS. 11A-11E.)

Humoral immune responses to SARS and MERS spike protein measured inINO-4800 treated rhesus monkeys. Day 0 and 28 intradermal delivery ofpDNA. Serum IgG binding ELISA. (FIGS. 12A-12G.; left panel, 1 mgINO-4800; right panel, 2 mg INO-4800).

Cellular immune responses measured by PBMC IFN-γ ELISpot inINO-4800-treated in rhesus monkeys following intradermal delivery ofpDNA on days 0 and 28. Results are shown in FIG. 13A (SARS CoV-2 Spikepeptides); 13B (SARS CoV Spike peptides); and 13C (MERS CoV Spikepeptides).

Example 4 INO-4800 SARS-CoV-2 Spike ELISA Assay

The SARS-CoV-2 spike protein is coated onto wells of a 96-wellmicroplate by incubating over night or for up to three days. Blockingbuffer is then added to block remaining free binding sites. Human serumsamples containing antibodies to SARS-COV-2 spike protein and assaycontrols are added to the blocked plate and incubated for 1 hour. Duringthe incubation, anti-spike protein antibodies present in the samples andpositive controls bind to spike protein immobilized onto the plate.Plates are then washed to remove unbound serum components. Next, ahorseradish peroxidase (HRP) labeled anti-human IgG antibody is added toallow for detection of antibody bound to the spike protein. After a onehour incubation, plates are washed to remove unbound HRP detectionantibody, and TMB substrate is added to plates. In the presence ofhorseradish peroxidase, the TMB substrate turns deep blue, proportionalto the amount of HRP present in the well. After allowing the reaction toproceed for approximately 10 minutes, an acid-based stop solution isadded, which halts the enzymatic reaction and turns the TMB yellow. Theyellow color is proportional to the amount of bound anti-spike proteinantibodies in each well and is read at 450 nm. The magnitude of theassay response is expressed as titers. Titer values are defined as thegreatest serial dilution at which the assay signal is greater than acutoff value based on the assay background levels for a panel of serumfrom normal human donors.

ELISA Assay Method Qualification

The INO-4800 SARS-CoV-2 Spike ELISA assay has been qualified and hasbeen found suitable for the its intended use to measure the humoralresponse in subjects participating in clinical trials involvingINO-4800. The formal qualification consisted of 18 plates and wasconducted by two operators over the course of four days. Thequalification determined the assay sensitivity, specificity,selectivity, and precision. At the time the assay was developedconvalescent sera was not available. A monoclonal antibody was thereforeused in development. The monoclonal antibody diluted in normal humansera was used to test all parameters in this assay. The overall assaysensitivity was found to be 16.1 ng/mL for 1/20-diluted serum, which is322 ng/mL for undiluted serum. Specificity was assessed bypre-incubating anti-spike protein antibody with recombinant spikeprotein prior to assay. Preincubation with the recombinant spike proteinresulted in greater than 60% signal reduction, indicating that theantibody was binding specifically to the spike protein coated to theplate and not to a different assay component. Selectivity wasinvestigated by spiking individual human serum samples with positivecontrol anti-spike antibody at a concentration near the limit ofdetection. Seven out of 10 individuals had signal above the cutoff, andeight out of the ten individuals had assay signal within 20% of the meansignal for the ten individuals, demonstrating that matrix effects areexpected to be minor for most human serum samples when diluted 1/20.Assay precision was assessed by assaying a high, low, and mediumanti-spike protein antibody positive control six times on each of sixplates. Results indicated low intra-assay raw signal variation but highraw signal inter-assay variation. Since each individual plate cutoff isbased on the signal of negative controls on each plate, inter-assayvariation in raw signal is not expected to influence the precision offinal titer calculations. To test this, the precision of plate cutoffswas evaluated in this qualification by titering the HPC (high positivecontrol) six times on each of six plates for a total of thirty-six titerevaluations. Thirty-five out of the thirty-six values were identical(titer of 180), while one of the titer determinations was one step lowerthan the rest (60 instead of 180). This resulted in an inter-assay CV of4.6%.

Example 5 INO-4800 SARS-CoV-2 Spike ELISPOT Assay

The enzyme-linked immunospot (ELISPOT) assay is a highly sensitiveimmunoassay that measures the frequency of cytokine-secreting cells atthe single-cell level. In this assay, cells are cultured on a surfacecoated with a specific capture antibody in the presence or absence ofstimuli. After an appropriate incubation time, cells are removed and thesecreted molecule is detected using a detection antibody in a similarprocedure to that employed by the ELISA. The detection antibody isbiotinylated and followed by a streptavidin-enzyme conjugate. By using asubstrate with a precipitating rather than a soluble product, the endresult is visible spots on the surface. Each spot corresponds to anindividual cytokine-secreting cell. The IFN-γ ELISPOT assayqualification was successfully completed with an assessment of assayspecificity, reproducibility and precision (intra-assay precision andinter-assay precision), dynamic range, linearity, relative accuracy,limit of detection and quantitation and assay robustness. The assay hasbeen tested and qualified under GLP/GCLP laboratory guidelines.

ELISPOT Assay Method Qualification. Specificity readings gave a meanvalue of <10 spot-forming units (SFU) for the assay negative control(medium with DMSO), a mean of 565 SFU for the positive control peptidepool CEF and a mean of 593 SFU in response to stimulation with mitogen(Phorbol Myristate Acetate+lonomycin). The highest reported % CV forintra-assay variation was 7.37%. The highest reported % CV forinter-assay variation was 17.23%. The highest observed % CV forinter-operator variability was 8.11%. These values fall below theFDA-recommended standard acceptance criteria of 20%.

Linearity of the dilution curve was demonstrated with a slope of 0.15and an R2 value of 0.99. Assay accuracy was >90% over the listed dynamicrange (156-5000 cells/well), falling within the acceptance criteria of80-120%. Limit of detection was determined to be 11 SFU/1×10⁶ PBMCs,limit of quantitation was observed at 20 SFU/1×10⁶ PBMCs. Robustness ofthe assay was evaluated by varying (i) peptide concentration; (ii)secondary antibody concentration; (iii) incubation times, and (iv)drying-out of plate membranes.

Based on the results of this qualification, the IFN-γ ELISPOT isconsidered qualified and ready for use in clinical trials.

Example 6 Phase 1 Open-Label Study to Evaluate the Safety, Tolerabilityand Immunogenicity of INO-4800, a Prophylactic Vaccine AgainstSARS-CoV-2, Administered Intradermally Followed by Electroporation inHealthy Volunteers

This is a Phase 1, open-label, multi-center trial (clinicaltrialsgovidentifier NCT04336410) to evaluate the safety, tolerability andimmunological profile of INO-4800 (pGX9501) administered by intradermal(ID) injection followed by electroporation (EP) using CELLECTRA® 2000device in healthy adult volunteers. Approximately 40 healthy volunteerswill be evaluated across two (2) dose levels: Study Group 1 and StudyGroup 2 as shown in Table 2. A total of 20 subjects will be enrolledinto each Study Group.

TABLE 2 COVID 19-001 Base Study Dose Groups Number Number of INO-4800IN0-4800 Total Dose of Study of Dosing Injections + EP (mg) per (mg) perINO-4800 Group Subjects Weeks per Dosing Visit injection Dosing Visit(mg) 1 20 0, 4 1 1.0 1.0 2.0 2 20 0, 4  2^(a) 1.0 2.0 4.0 Total 40^(a)INO-4800 will be injected ID followed by EP in an acceptablelocation on two different limbs at each dosing visitAll subjects are followed for 24 weeks following the last dose. Week 28is the End of Study (EOS) visit.

Primary Objectives:

-   -   Evaluate the tolerability and safety of INO-4800 administered by        ID injection followed by EP in healthy adult volunteers    -   Evaluate the cellular and humoral immune response to INO-4800        administered by ID injection followed by EP

Primary Safety Endpoints:

-   -   Incidence of adverse events by system organ class (SOC),        preferred term (PT), severity and relationship to        investigational product    -   Administration (i.e., injection) site reactions (described by        frequency and severity)    -   Incidence of adverse events of special interest

Primary Immunogenicity Endpoints:

-   -   SARS-CoV-2 Spike glycoprotein antigen-specific antibodies by        binding assays    -   Antigen-specific cellular immune response by IFN-        , ELISpot and/or flow cytometry assays

Exploratory Objective:

-   -   Evaluate the expanded immunological profile by assessing both T        and B cell immune response

Exploratory Endpoint:

-   -   Expanded immunological profile which may include (but not        limited to) additional assessment of T and B cell numbers,        neutralization response and T and B cell molecular changes by        measuring immunologic proteins and mRNA levels of genes of        interest at all weeks as determined by sample availability

Safety Assessment:

Subjects are followed for safety for the duration of the trial throughthe end of study (EOS) or the subject's last visit. Adverse events arecollected at every visit (and a Day 1 phone call). Laboratory blood andurine samples are drawn at Screening, Day 0 (pregnancy test only), Week1, Week 4 (pregnancy test only), Week 6, Week 8, Week 12 and Week 28,according to the Schedule of Events (Table 3). All adverse events,regardless of relationship, are collected from the time of consent untilEOS. All serious adverse events, adverse events of special interest andtreatment-related adverse events are followed to resolution orstabilization.

TABLE 3 Clinical Trial Schedule of Events Week 4 Day 0 Day 1 Week 1 (±5d) Week 6 Week 8 Week 12 Week 28 Tests and assessments Screen^(a) PrePost (+1 d) (±3 d) Pre Post (±5 d) (±5 d) (±5 d) (±5 d) Informed ConsentX Inclusion/Exclusion X Criteria Medical History X X Demographics XConcomitant Medications X X X X X X X X Physical Exam^(b) X X X X X X XX Vital Signs X X X X X X X X Height and Weight X CBC with DifferentialX X X X X X Chemistry^(c) X X X X X X Serology^(d) X 12-lead ECG XUrinalysis Routine^(e) X X X X X X Pregnancy Test^(f) X X X INO-4800 +EP^(g)  X^(h)  X^(h) Download EP Data^(i) X X Adverse Events^(j) X X XX^(k) X X X X X X X Immunology (Whole X X X X X X X blood)^(l)Immunology (Serum)^(m) X X X X X X X ^(a)Screening assessment occursfrom −30 days to −1 day prior to Day 0. ^(b)Full physical examination atscreening and Week 28 (or any other study discontinuation visit) only.Targeted physical exam at all other visits. ^(c)Includes Na, K, Cl,HCO3, Ca, PO4, glucose, BUN, and Cr. ^(d)HIV antibody or rapid test,HBsAg, HCV antibody. ^(e)Dipstick for glucose, protein, and hematuria.Microscopic examination should be performed if dipstick is abnormal.^(f)Serum pregnancy test at screening. Urine pregnancy test at othervisits. ^(g)All doses delivered via intradermal injection followed byEP. ^(h)For Study Group 1, one injection in skin preferably over deltiodmuscle at Day 0 and Week 4. For Study Group 2, two injections in skinwith each injection over a different deltoid or lateral quadriceps;preferably over the deltoid muscles, at Day 0 and Week 4. ^(i)Followingadministration of INO-4800, EP data will be downloaded from theCELLECTRA ® 2000 device and provided to Inovio. ^(j)Includes AEs fromthe time of consent and all injection site reactions that qualify as anAE. ^(k)Follow-up phone call to collect AEs. ^(l)4 × 8.5 mL (34 mL)whole blood in 10 mL Acid Citrate Dextrose (ACD, Yellow top) tubes pertime point. Note: Collect a total of 68 mL whole blood prior to 1st dose(screening and prior to Day 0 dosing). ^(m)1 × 8 mL blood in 10 mL redtop serum collection tube per time point. Note: Collect four aliquots of1 mL each (total 4 mL) serum at each time point prior to 1st dose(Screening and prior to Day 0 dosing).

Immunogenicity Assessment:

Immunology blood samples are collected at Screening, Day 0 (prior todose), Week 4 (prior to dose), Week 6, Week 8, Week 12 and Week 28.Determination of analysis of collected samples for immunologicalendpoints are determined on an ongoing basis throughout the study.

Clinical Trial Population:

Healthy adult volunteers between the ages of 18-50 years, inclusive.

Inclusion Criteria:

-   -   a. Adults aged 18 to 50 years, inclusive;    -   b. Judged to be healthy by the Investigator on the basis of        medical history, physical examination and vital signs performed        at Screening;    -   c. Able and willing to comply with all study procedures;    -   d. Screening laboratory results within normal limits or deemed        not clinically significant by the Investigator;    -   e. Negative serological tests for Hepatitis B surface antigen        (HBsAg), Hepatitis C antibody and Human Immunodeficiency Virus        (HIV) antibody screening;    -   f. Screening electrocardiogram (ECG) deemed by the Investigator        as having no clinically significant findings (e.g.        Wolff-Parkinson-White syndrome);    -   g. Use of medically effective contraception with a failure rate        of <1% per year when used consistently and correctly from        screening until 3 months following last dose, be        post-menopausal, be surgically sterile or have a partner who is        sterile.

Exclusion Criteria:

-   -   a. Pregnant or breastfeeding, or intending to become pregnant or        father children within the projected duration of the trial        starting with the screening visit until 3 months following last        dose;    -   b. Is currently participating in or has participated in a study        with an investigational product within 30 days preceding Day 0;    -   c. Previous exposure to SARS-CoV-2 (laboratory testing at the        Investigator's discretion) or receipt of an investigational        vaccine product for prevention of COVID-19, MERS or SARS;    -   d. Current or history of the following medical conditions:        -   Respiratory diseases (e.g., asthma, chronic obstructive            pulmonary disease);        -   Hypertension, sitting systolic blood pressure >150 mm Hg or            a diastolic blood pressure >95 mm Hg;        -   Malignancy within 5 years of screening;        -   Cardiovascular diseases (e.g., myocardial infarction,            congestive heart failure, cardiomyopathy or clinically            significant arrhythmias);    -   e. Immunosuppression as a result of underlying illness or        treatment including:        -   Primary immunodeficiencies;        -   Long term use (≥7 days) of oral or parenteral            glucocorticoids;        -   Current or anticipated use of disease modifying doses of            anti-rheumatic drugs and biologic disease modifying drugs;        -   History of solid organ or bone marrow transplantation;        -   Prior history of other clinically significant            immunosuppressive or clinically diagnosed autoimmune            disease.    -   f. Fewer than two acceptable sites available for ID injection        and EP considering the deltoid and anterolateral quadriceps        muscles;    -   g. Any physical examination findings and/or history of any        illness that, in the opinion of the study investigator, might        confound the results of the study or pose an additional risk to        the patient by their participation in the study.

Clinical Trial Treatment:

The INO-4800 drug product contains 10 mg/mL of the DNA plasmid pGX9501in 1×SSC buffer (150 mM sodium chloride and 15 mM sodium citrate). Avolume of 0.4 mL is filled into 2-mL glass vials that are fitted withrubber stoppers and sealed aluminum caps. INO-4800 is stored at 2-8° C.

Study Group 1 is administered one 1.0 milligram (mg) intradermal (ID)injection of INO-4800 followed by electroporation (EP) using theCELLECTRA® 2000 device per dosing visit at Day 0 and Week 4. Study Group2 is administered two 1.0 mg ID injections (total 2.0 mg per dosingvisit) (in an acceptable location on two different limbs) of INO-4800followed by EP using the CELLECTRA® 2000 device at Day 0 and Week 4.

Peripheral Blood Immunogenicity Assessments

Whole blood and serum samples are obtained. Immunology blood and serumsamples are collected at Screening and at visits specified in theSchedule of Events (Table 2). Both Screening and Day 0 immunologysamples are required to enable all immunology testing. The T and B cellimmune responses to INO-4800 are measured using assays that may includebut are not limited to ELISA, neutralization, assessment ofimmunological gene expression, assessment of immunological proteinexpression, flow cytometry and ELISPOT. The ELISA binding assay is astandard plate-based ELISA using 96-well ELISA plates. Plates are coatedwith SARS-CoV-2 spike protein and blocked. Following blocking, sera fromvaccinated subjects are serially diluted and incubated on the plate. Asecondary antibody that is able to bind human IgG is used to assess thelevel of vaccine specific antibodies in the sera. T-cell response isassessed by an IFN-gamma ELISPOT assay. PBMCs isolated from studyvolunteers are incubated with peptide fragments of the SARS-CoV-2 spikeprotein. The cells and peptides are placed in a MabTech plates coatedwith an antibody that captures IFN-gamma. Following 24 hours ofstimulation, cells are washed out and a secondary antibody that bindsIFN-gamma is added. Each vaccine specific cell creates a spot that canbe counted to determine the level of cellular responses induced. Inaddition, humoral responses to SARS-CoV-2 Nucleocapsid Protein (NP) mayalso be assessed to rule out potential infection by wild-type SARS-CoV-2post INO-4800 treatment during the study. Determination of analysis ofcollected samples for immunological endpoints is determined on anongoing basis throughout the study.

Primary Outcome Measure:

-   -   1. Percentage of Participants with Adverse Events (AEs) [Time        Frame: Baseline up to Week 28]    -   2. Percentage of Patients with Administration (Injection) Site        Reactions [Time Frame: Day 0 up to Week 28]    -   3. Incidence of Adverse Events of Special Interest (AESIs) [Time        Frame: Baseline up to Week 28]    -   4. Change from Baseline in Antigen-Specific Binding Antibody        Titers [Time Frame: Baseline up to Week 28] A subject is        considered to have a positive antibody response if the optical        density post vaccine is 2.0 SD higher than the optical density        at day 0 and above the ELISA specific cut off    -   5. Change from Baseline in Antigen-Specific Interferon-Gamma        (IFN-γ) Cellular Immune Response [Time Frame: Baseline up to        Week 28] A subject is considered to have a positive cellular        response if the number of IFN-gamma producing cells (spots) post        vaccine is 2.0 SD higher than the number of spots at day 0 and        above the assay LOD.

The safety of INO-4800 is measured and graded in accordance with the“Toxicity Grading Scale for Healthy Adult and Adolescent VolunteersEnrolled in Preventive Vaccine Clinical Trials”, issued September 2007(Appendix A). An adverse event of special interest (AESI) (serious ornon-serious) is one of scientific and medical concern specific to theproduct or program. AESIs include those listed in Table 4.

TABLE 4 Body System AESI Respiratory Acute respiratory distress syndrome(ARDS) Pneumonitis/Pneumonia Neurologic Generalized convulsion Asepticmeningitis Guillain-Barré Syndrome (GBS) Encephalitis/Myelitis Acutedisseminated encephalomyelitis (ADEM) CNS vasculopathy (stroke)Hematologic Thrombocytopenia Disseminated intravascular coagulation(DIC) Immunologic Anaphylaxis Vasculitides Enhanced disease followingimmunization Other Local/systemic SAEs Acute cardiac injury Acute kidneyinjury Septic shock-like syndrome

Dose Limiting Toxicity (DLT)

For the purpose of this clinical trial, the following are dose limitingtoxicities:

-   -   Grade 3 or greater local injection site erythema, swelling        and/or induration observed ≥1 day after INO-4800 administration        (see Table 5);    -   Pain or tenderness at the injection site that requires        hospitalization despite proper use of non-narcotic analgesics;    -   Grade 4 or greater non-injection site adverse event assessed by        the PI as related to INO-4800 administration;    -   Grade 4 or greater clinically significant laboratory        abnormalities assessed by the PI as related to INO-4800        administration.

TABLE 5 Grading Scale for Injection Site Reactions Local ReactionPotentially to Life Injectable Threat- Product Mild Moderate Severeening (Grade) (1) (2) (3) (4) Pain Does not Repeated Any use Emergencyinterfere use of non- of narcotic room with narcotic pain visit oractivity pain reliever hospital- reliever or ization >24 prevents hoursor daily interferes activity with activity Tender- Mild DiscomfortSignificant ER visit or ness discomfort with discomfort hospital- totouch movement at rest ization Erythema/ 2.5-5 cm 5.1-10 >10 cm Necrosisor Rednessª cm exfoliative dermatitis Induration/ 2.5-5 cm 5.1-10 >10 cmor Necrosis Swelling^(b) and no cm or prevents inter- interferes dailyference with activity w/activity activity September 2007 “FDA Guidancefor Industry-Toxicity Grading Scale for Healthy Adult and AdolescentVolunteers Enrolled in Preventive Vaccine Clinical Trials” ^(a)Inaddition to grading the measured local reaction at the greatest singlediameter, the measurement should be recorded as a continuous variable^(b)Should be evaluated and graded using the functional scale as well asthe actual measurement.

Analytical Populations

Analysis populations are:

-   -   The modified intention to treat (mITT) population includes all        subjects who receive at least one dose of the INO-4800. Subjects        in this sample are analyzed by their assigned dose group of        INO-4800. The mITT population is used to analyze co-primary and        exploratory immunological endpoints.    -   The per-protocol (PP) population is comprised of mITT subjects        who receive all their planned administrations and who have no        Medical Monitor-assessed important protocol violations. Analyses        on the PP population is considered supportive of the        corresponding mITT analyses.

The safety analysis population includes all subjects who receive atleast one dose of INO 4800 administered by ID injection. Subjects forthis population are grouped in accordance with the dose of INO-4800 thatthey received. This population is used for all safety analyses in thestudy.

Primary Safety Analyses

The primary analyses for this trial are safety analyses of treatmentemergent adverse events (TEAEs), administration site reactions andclinically significant changes in safety laboratory parameters frombaseline.

TEAEs are defined for this trial as any adverse events, adverse eventsof special interest, or serious adverse events that occur on or afterDay 0 following IP administration. All TEAEs are summarized byfrequency, percentage and associated 95% Clopper-Pearson confidenceinterval. The frequencies are presented separately by dose number andare depicted by system order class and preferred term. Additionalfrequencies are presented with respect to maximum severity andrelationship to IP. Multiple occurrences of the same AE in a singlesubject are counted only once following a worst-case approach withrespect to severity and relationship to IP. All serious TEAEs aresummarized as above. AE duration is calculated as AE stop date—AE startdate +1 day. AEs and SAEs that are not TEAEs or serious TEAEs arepresented in listings.

All of these primary safety analyses are conducted on the subjects inthe safety population.

Primary Immunogenicity Analyses

SARS-CoV-2 Spike glycoprotein antigen specific binding antibody titers,and specific cellular immune responses are analyzed by Study Groupwithin age strata. Binding antibody titer is analyzed for each StudyGroup using the geometric mean and associated 95% confidence intervals.Antigen specific cellular immune response increases are analyzed foreach Study Group using medians, inter-quartile range and 95% confidenceintervals. Change from baseline for both binding antibody titer andantigen specific cellular response increases are analyzed usingGeometric Mean Fold Rise and 95% confidence intervals. Binding antibodytiters are analyzed between each Study Group pair within age stratausing the geometric mean ratio and associated 95% confidence intervals.Antigen specific cellular immune responses are analyzed between eachStudy Group pair within age strata using median differences andassociated 95% confidence intervals. All of these primary immunogenicityanalyses are conducted on the subjects in the mITT and PP populations.

Exploratory Analyses

T and B post baseline cell number will be analyzed descriptively byStudy Group with means/medians and associated 95% confidence intervals.Percent neutralizing antibodies will be analyzed for each Study Groupusing medians, inter-quartile range and 95% confidence intervals.

The safety and immunogenicity of the optional booster dose of INO-4800following a prior two-dose regimen will be analyzed as described below.Live neutralization reciprocal antibody titer and pseudoneutralizationreciprocal antibody titer will be analyzed for each Study Group withinage strata using the geometric mean and associated 95% confidenceintervals. Fold rise from baseline will tabulated for each immunogenicbiomarker. If there is sufficient data for analysis, exploratory betweengroup immunogenic comparisons between subjects who opt for just 2administrations and subjects who opt for 2 administrations plus thebooster administration will be undertaken.

Further exploration of the effect of age and other potential confounderson the relationship between immune biomarkers and INO-4800 dose mayinvolve the use of ANCOVA and/or Logistic regression models.

Preliminary Base Study Results

All 8 adverse events reported were Grade 1; 5 due to local injectionsite reactions. No serious adverse events, adverse events of specialinterest, or dose limiting toxicities were reported.

Preliminary Binding ELISA Analysis demonstrated 7/9 (78%) subjects hadpositive antibody responses. Responders had a four-fold increase intiter.

At week six, multiple immunology assays, including those for humoral andcellular immune response, were conducted for both 1.0 mg and 2.0 mg dosecohorts after two doses. Analyses at that point showed that 94% (34 outof 36 total trial participants) demonstrated overall immunologicalresponse rates based on preliminary data assessing humoral (binding andneutralizing) and T cell immune responses. One participant in the 1 mgdose cohort and two participants in the 2 mg dose cohort were excludedfrom the immune analyses as they tested positive for COVID-19 immuneresponses at study entry, indicating prior infection. One participant inthe 2 mg dose cohort discontinued the study for reasons unrelated tosafety or tolerability.

Through week eight, INO-4800 was generally safe and well-tolerated inall participants in both cohorts. All ten reported adverse events (AEs)were grade 1 in severity, with most being injection site redness. Therewere no reported serious adverse events (SAEs).

Initial Phase I Results

Study Population Demographics

A total of 55 participants were screened and 40 participants wereenrolled into the initial two groups (FIG. 16 ). The median age was 34.5years (range 18 to 50 years). Participants were 55% male (Table 6). Mostparticipants were white (82.5%).

TABLE 6 Group 1, Group 2, 1 mg 2 mg Overall Variable Statistic (N = 20)(N-20) (N = 40) Gender Male n (%) 11 (55.0) 11 (55.0) 22 (55.0) Female n(%)  9 (45.0)  9 (45.0) 18 (45.0) Race White n (%) 18 (90.0) 15 (75.0)33 (82.5) Black or n (%) 1 (5.0) 1 (5.0) 2 (5.0) African American Asiann (%) 1 (5.0)  4 (20.0)  5 (12.5) Ethnicity Hispanic n (%) 0 0 0 orLatino Not n (%)  20 (100.0)  20 (100.0)  40 (100.0) Hispanic or LatinoAge n 20 20 40 (years) Mean 35.0 35.6 35.3 (SD) (10.69) (9.18) (9.84)Median 33.0 38.0 34.5 Min, Max 18, 50 19, 50 18, 50 Baseline n 20 19 39Height (cm) Mean 172.59 172.16 172.38 (SD) (10.853) (8.631) (9.707)Median 169.75 170.10 170.10 Min, Max 155.9, 195.6 158.0, 188.0 155.9,195.6 Baseline n 20 19 39 Weight (kg) Mean 74.13 71.35 72.77 (SD)(14.701) (12.611) (13.615) Median 70.45 69.00 69.60 Min, Max  58.5,110.0 55.0, 92.5  55.0, 110.0

The vaccine was administered in 0.1 ml intradermal injections followedby EP at the site of vaccination. EP was performed using CELLECTRA® 2000with four 52-msec pulses at 0.2A (40 to 200 V, depending on tissueresistance) per season. The first two pulses were spaced 0.2 secondsapart followed by a 3-second pause before the final two pulses that werealso spaced by 0.2 seconds. The dose groups were enrolled sequentiallywith a safety run-in for each. Participants were and will be evaluatedclinically and for safety on Day 1 and at Weeks 1, 4 (Dose 2), 6, 8, 12,28, 40 and 52. Safety laboratory testing (complete blood count,comprehensive metabolic panel and urinalysis) were and will be conductedon all follow-up visits except for Day 0, Day 1 and Week 4. Immunologyspecimens were obtained at all time points post-dose 1 except Day 1 andWeek 1. Local and systemic AEs, regardless of relationship to thevaccine, were recorded and graded by the investigator. AEs were gradedaccording to the Toxicity Grading Scale for Healthy Adult and AdolescentVolunteers Enrolled in Preventive Vaccine Clinical Trials guidelinesthat were issued by the Food and Drug Administration in September 2007.

Vaccine Safety and Tolerability

39 (97.5%) completed both doses and 1 subject in the 2.0 mg groupdiscontinued trial participation prior to receiving the second dose dueto lack of transportation to the clinical sites, unrelated to the studyor the dosing. All 39 remaining subjects completed the visit 8 weekspost-dose 1. There were a total of 11 local and systemic AEs reported by8 weeks post-dose 1, six of these were deemed related to vaccine. AllAEs were mild or Grade 1 in severity. The most frequent AEs wereinjection site reactions including injection site pain (3) and erythema(2). One systemic AE related to the vaccine was nausea. There were nofebrile reactions. No subjects discontinued the trial due to an AE. Noserious adverse events (SAEs) nor AESIs were reported. There were noabnormal laboratory values of clinical concern throughout the initial8-week follow-up period. There was no increase in the number ofparticipants who experienced AEs related to the vaccine in the 2.0 mggroup (10% of subjects), compared to that in the 1.0 mg group (15% ofsubjects). In addition, there was no increase in frequencies of AEs withthe second dose over the first dose in both dose level groups. TheINO-4800 Phase 1 safety data thus suggests that the vaccine is likely asafe booster as there was no increase frequency of side effects afterthe second vaccine administration compared to the first dose.

Immunogenicity: Thirty-eight subjects were included in theimmunogenicity analysis. In addition to one subject in the 2.0 mg groupwho discontinued prior to completing dosing, one subject in the 1.0 mggroup was deemed seropositive at baseline and was excluded.

Humoral Immune Responses: Serum samples were used to measureneutralizing antibody titers against SARS-CoV-2/Australia/VIC01/2020isolate and binding antibodies to RBD and whole spike 51+S2 protein.

S1+S2 Enzyme-Linked Immunosorbent Assay (ELISA): A standard bindingELISA was used to detect serum binding anti-SARS-CoV-2 spike antibodies.ELISA plates were coated with recombinant S1+S2 SARS-CoV-2 spike protein(Sino Biological) and incubated overnight and blocked. Samples wereserially diluted and incubated on the blocked assay plates for one hour.The magnitude of the assay response was expressed as titers which weredefined as the greatest serial dilution at which the optical density 3SD above background Day 0. 68% of participants in the 1.0 mg group and70% of participants in the 2.0 mg group had at least an increase inserum IgG binding titers to S1+S2 spike protein when compared to theirpre-vaccination time point (Day 0), with the responder GMT of 320.0 (95%CI: 160.5, 638.1) and 508.0 (95% CI: 243.6, 1059.4) in the 1.0 mg and2.0 mg groups, respectively (FIG. 17C). In FIG. 17D, the humoralresponse in the 1.0 mg dose group and 2.0 mg dose group was assessed forthe ability to bind whole spike protein (51 and S2) (n=19, 1.0 mg; n=19,2.0 mg). End point titers were calculated as the titer that exhibited anOD 3.0 SD above baseline, titers at baseline were set at 1. A responseto live virus neutralization was a PRNT IC50≥10. In all graphshorizontal lines represent the Median and bars represent theInterquartile Range.

Sera was also tested for the ability to neutralize live virus inSARS-CoV-2 wildtype virus neutralization assays.SARS-CoV-2/Australia/VIC01/2020 isolate neutralization assays wereperformed at Public Health England (Porton Down, UK). Neutralizing virustiters were measured in serum samples that had been heat-inactivated at56° C. for 30 min. SARS-CoV-2 (Australia/VIC01/2020 isolate44) wasdiluted to a concentration of 933 pfu ml-l and mixed 50:50 in 1% FCS/MEMcontaining 25 mM HEPES buffer with doubling serum dilutions. After 5days incubation at 37° C. in a humidified box, the plates were fixed,stained and plaques counted. Virus titer were determined using astandard 50% tissue culture infection dose (TCID50) assay. After thesecond vaccination at week 6, the responder geometric mean titer (GMT)by live virus PRNT IC50 neutralization assay were 82.4 and 63.5 in the1.0 mg and 2.0 mg groups, respectively. The percentage of responders(post vaccination PRNT IC50 ≥10) were 83% and 84% in the 1.0 mg and 2.0mg groups, respectively (FIG. 17A and Table 7).

TABLE 7 Live SARS-CoV-2 Neutralization 1.0 mg 2.0 mg N = 18* N = 19Overall Week 6 GMT Reciprocal 44.4 34.9 Titer (95% CI) (14.6, 134.8)(15.8, 77.2) Range 1, 11647  1, 652 Responders** n (%) 15 (83%) 16 (84%)Week 6 GMT Reciprocal 82.4 63.5 Titer (95% CI) (29.1, 233.3) (39.6,101.8) Range 4, 11647 13, 652 *Excludes one subject with baselinepositive NP ELISA **Week 6 PRNT IC₅₀ ≥10, or ≥4 if binding ELISAactivity is seen

RBD Enzyme-Linked Immunosorbent Assay (ELISA): MaxiSorp 96-well plates(ThermoFisher, 439454) were coated with 50 ul/well of 1 ug/ml ofSARS-CoV-2 RBD (SinoBiological, 40592-VO8H), protein diluted in PBS andincubated at 4° C. overnight. Plates were washed 4 times with PBST (PBSwith 0.05% Tween-20) and blocked with 200 ul/well of blocking buffer(PBS with 5% non-fat dry milk and 0.1% Tween-20) at room temperature for2 hr. After washing with PBST, 50 ul/well of sera sample seriallydiluted in blocking buffer was added to the plate in duplicate andincubated at room temperature for 2 hr. After washing with PBST, 50ul/well of anti-human-IgG-HRP detection antibody (BD Pharmingen, 555788)diluted 500-fold in blocking buffer was added and incubated at roomtemperature for 1 hr. After washing with PBST, 50 ul/well of 1-StepUltra TMB (Thermo, 34028) was added and incubated at room temperaturefor 5 min. 50 ul/well of 2M sulfuric acid was added to stop the colorchange reaction and optical absorbance was measured at 450 and 570 nm ona Synergy 2 microplate reader (Biotek). Endpoint titers were defined asthe greatest serial dilution at which the OD450-570 values were 3standard deviations above the matched Day 0 signal. At week 6, theresponder GMT were 385.6 (95% CI: 69.0, 2154.9) and 222.1 (95% CI: 87.0,566.8) in the 1.0 mg and 2.0 mg groups, respectively (FIG. 17B).

Overall seroconversion (defined as those participants who respond withneutralization or binding antibodies to S protein or RBD) after 2vaccine doses in 1.0 mg and 2.0 mg dose group were 89% and 95%,respectively.

Cellular Responses: Peripheral Blood Mononuclear Cells (PBMCs) wereisolated from blood samples, frozen and stored in liquid nitrogen forsubsequent analyses.

INO-4800 SARS-CoV-2 Spike ELISPOT. Peripheral mononuclear cells (PBMCs)were isolated pre- and post-vaccination. Cells were stimulated in vitrowith a pool of 15-mer peptides (overlapping by 9 residues) spanning thefull-length consensus spike protein sequence. Cells were incubatedovernight (18-22h, 37C, 5% CO2) with peptide pools (225 μg/ml), DMSOalone (0.5%, negative control) or PMA and Ionomycin (positive controls).The next day, cells were washed off, and the plates were developed: Thedetection antibody is biotinylated and followed by a streptavidin-enzymeconjugate. By using a substrate with a precipitating rather than asoluble product, resulting in visible spots. Each spot corresponds to anindividual cytokine-secreting cell. After plates were developed, spotswere scanned and quantified using the CTL S6 Micro Analyzer (CTL) withImmunoCapture™ and ImmunoSpot™ software. Values are shown asbackground-subtracted average of measured triplicates.

The percentage of responders at week 8 was 74% in the 1.0 mg dose group,and 100% in the 2.0 mg dose group (Table 8). The Median SFU per 106 PBMCwas 46 and 71 for the responders in 1.0 mg and 2.0 mg dose groups,respectively. In each group, there were statistically significantincreases in the numbers of interferon-γ-secreting cells (SFU) obtainedper million PBMCs over baseline (P=0.001 and P<0.0001, respectively,Wilcoxon matched-pairs signed rank test, post-hoc analysis), FIG. 18A.Interestingly, 5 non-responders in 1.0 mg group by T cell ELISPot assayshowed strong reactivity by live virus neutralization assay. It is alsointeresting to note that 3 convalescent samples tested by the ELISpotassay showed lower T cell responses, with a median of 33, than the 2.0mg dose group at Week 8. INO-4800 generated strong T cell responses thatwere more frequent and a higher responder median response (45.6 vs 71.1)in the 2.0 mg dose group. The 2.0 mg group's T cell responses weremapped to 5 epitope pools as shown in FIG. 18B. Interestingly T cellresponses in the all regions of the Spike protein were observed.

TABLE 8 Immune Responses 1.0 mg 2.0 mg Cohort Re- Cohort Re- Outputsponders^(‡) Output sponders^(‡) Immune Assay Value n (%) Value n (%)Neutralization 44.4 15/18 34.9 16/19 Week 6 GMT [14.6, (83%)   [15.8,(84%)  Reciprocal Titer 134.8] 77.2] [95% CI] (Range) (1, (1, 652)11647) RBD Binding 27.3 10/18 66.8 14/18 Antibody Week 6 [4.8, (56%)  [17.4, (78%)  GMT Reciprocal 156.8] 257.5] Titer [95% CI] (1, (1, 3125)(Range) 15625) S1 + S2 Binding 174.4 17/19 136.8 15/19 Antibody Week 6[59.9, (89%)   [34.5, (79%)  GMT Reciprocal 507.3] 543.1] Titer [95% CI](1, (1, 2560) (Range) 2560) IFN-gamma 26.2 SFU 14/19 71 SFU 19/19ELISpot Week 8 [10-64] (74%) ^(μ) [32-194] (100%) ^(μ) Median SFU per(1, (8.9, [95% CI] (Range) 374.4) 615.6) 1.0 mg Cohort excludes onesubject with baseline ELISA titer of 1280 ^(‡)Response criteria:Neutralization-Week 6 PRNT IC₅₀ ≥10, or 24 if binding ELISA activity isseen RBD Binding-Week 6 value >1 ELISpot-Value ≥12 SFU over Week 0 ^(μ)Responders generated using Week 6 and Week 8 data

INO-4800 SARS-CoV-2 Spike Flow Cytometry Assay: The contribution of CD4+and CD8+ T cells to the cellular immune response against INO-4800 wasassessed by intracellular cytokine staining (ICS). PBMCs were also usedfor Intracellular Cytokine Staining (ICS) analysis using flow cytometry.One million PMBCs in 200 uL complete RPMI media were stimulated for sixhours (37° C., 5% CO₂) with DMSO (negative control), PMA and Ionomycin(positive control, 100 ng/mL and 2 μg/mL, respectively), or with theindicated peptide pools (225 μg/mL). After one hour of stimulation,Brefeldin A and Monensin (BD GolgiStop and GolgiPlug, 0.001% and0.0015%, respectively) were added to block secretion of expressedcytokines. After stimulation the cells were moved to 4° C. overnight.Next, cells were washed in PBS for live/dead staining (Life TechnologiesLive/Dead aqua fixable viability dye, as previously described), and thenresuspended in FACS buffer (0.5% BSA, 2 mM EDTA, 20 mM HEPES). Next,cells were stained for extracellular markers, fixed and permeabilized,and then stained for the indicated cytokines (Table 9) for antibodiesused for flow cytometry.

TABLE 9 Flow Cytometry Panel Tube Channel Marker/Cytokine  1 UnstainedNA  2 BV510 Live/Dead Fix Aqua  3 BUV737 CD8  4 APC-Cy7 IL-2  5 BV650CD45RA  6 APC CD3  7 BV786 CD14/CD16/CD19  8 BV711 IFN-gamma  9 BV421CCR7 10 PE-Cy7 IL-17 11 FITC FITC 12 PE Dazzle (PE-CF594) IL-4 13 PE CD107a 14 PerCP-eFluor710 (PerCP-Cy5.5) CD4

CD8+ T cells producing IFN-

, TNF-α and/or IL-2 (any response) were statistically significantlyincreased post vaccination in the 2.0 mg dose group (FIG. 18C, P=0.0181,Wilcoxon matched-pairs signed rank test, post-hoc analysis). CD4+ Tcells producing TNF-α were also statistically significantly increased inthe 2.0 mg dose group (FIG. 18C, P=0.0020, Wilcoxon matched-pairs signedrank test, post-hoc analysis).

CD4+ and CD8+ T cells were explored following vaccination. Nearly half(47%) of the CD8+ T cells in the 2.0 mg dose group were dual producingIFN-

and TNF-α (FIG. 18E). CD8+ T cells producing cytokine in the 1.0 mg dosegroup were primarily monofunctional IFN-

producing cells. The CD4+ T cell compartment was highly polyfunctionalwith 6% and 9% (in the 1.0 mg and 2.0 mg dose groups, respectively)producing all 3 cytokines, IFN-

, TNF-α, and IL-2.

The composition of CD4+ or CD8+ T cells producing any cytokine (anyresponse, IFN-

or TNF-α or IL-2 following vaccination) was also assessed for surfacemarkers CCR7 and CD45RA to characterize effector (CCR7−CD45RA+),effector memory (CCR7−CD45RA−), and central memory (CCR7+CD45RA−) cells(FIG. 18D). In both dose groups, CD8+T cells making cytokine in responseto stimulation with spike peptides were balanced across the threepopulations, whereas CD4+ T cells were predominantly of the centralmemory phenotype (FIG. 18D).

Th2 responses were also measured by assessing IL-4 production, and nostatistically significant increases (Wilcoxon matched-pairs signed ranktest, post-hoc analysis) were observed in either group post vaccination(FIG. 18F).

In this Phase 1 trial, INO-4800 vaccination led to potent T cellresponses with increased Th1 phenotype, demonstrated by both IFN-

ELISpot as well as multiparametric flow cytometry, as evidenced byincreased expression of Th1-type cytokines IFN-

, TNF-α. and IL-2 (FIG. 18C). Assessment of polyfunctionality of T cellsinduced by INO-4800 suggested the presence of SARS-CoV-2 specific CD4+and CD8+ T cells exhibiting hallmarks of memory status suggest that apersistent cellular response has been established (FIG. 18D).Importantly, this was accomplished while minimizing induction of IL-4, aprototypical Th2 cytokine (FIG. 18F).

Phase 1 Update

This was designed as a Phase 1, open-label, multicenter trial(NCT04336410) to evaluate the safety, tolerability and immunogenicity ofINO-4800 administered intradermally (ID) followed by electroporationusing the CELLECTRA 2000 device. Healthy participants 18 to 50 years ofage without a known history of COVID-19 illness received either a 1.0 mgor 2.0 mg dose of INO-4800 in a 2-dose regimen (Weeks 0 and 4).

DNA vaccine INO-4800. The vaccine was produced according to current GoodManufacturing Practices. INO-4800 contains plasmid pGX9501 expressing asynthetic, optimized sequence of the SARS-CoV-2 full length spikeglycoprotein which was optimized as previously described at aconcentration of 10 mg/ml in a saline sodium citrate buffer.

Endpoints. Safety endpoints included systemic and local administrationsite reactions up to 8 weeks post-dose 1. Immunology endpoints includeantigen-specific binding antibody titers, neutralization titers andantigen-specific interferon-gamma (IFN-

) cellular immune responses after 2 doses of vaccine. For Live VirusNeutralization, a responder is defined as Week 6 PRNT IC50>10, or >4 ifa subject is a responder in ELISA. For S1+S2 ELISA, a responder isdefined as a Week 6 value >1. For the ELISpot assay, a responder isdefined as a Week 6 or Week 8 value that is >12 spot forming units per106 PBMCs above Week 0.

Study Procedures.

Forty participants were enrolled into two groups; 20 participants ineach of 1.0 mg and 2.0 mg dose groups that received their doses on Weeks0 and 4. The vaccine was administered in 0.1 ml intradermal injectionsin the arm followed by EP at the site of vaccination. Subjects in the1.0 mg dose group received one injection on each dosing visit. Thesecond dose of the vaccine could be injected in the same arm or adifferent arm relative to the first dose. Subjects in the 2.0 mg dosegroup received one injection in each arm at each dosing visit. EP wasperformed using CELLECTRA® 2000 as previously described. The devicedelivers total four electrical pulses, each 52 ms in duration atstrengths of 0.2 A current and voltage of 40-200 V per pulse. The dosegroups were enrolled sequentially with a safety run-in for each. The 1.0mg dose group enrolled a single participant per day for 3 days. Anindependent Data Safety Monitoring Board (DSMB) reviewed the Week 1safety data and based on a favorable safety assessment, made arecommendation to complete enrollment of the additional 17 participantsinto that dose group. In a similar fashion, the 2.0 mg dose group wassubsequently enrolled. Participants were assessed for safety andconcomitant medications at all time points, including screening, Week 0(Dose 1), post dose next day phone call, Week 1, 4 (dose 2), 6, 8, 12,28, 40 and 52 post-dose 1. Local and systemic AEs, regardless ofrelationship to the vaccine, were recorded and graded by theinvestigator. Safety laboratory testing (complete blood count,comprehensive metabolic panel and urinalysis) were and will continue tobe conducted at screening, Week 1, 6, 8, 12, 28 and 52 post-dose 1.Immunology specimens were obtained at all time points post-dose 1 exceptat Day 1 and Week 1. AEs were graded according to the Toxicity GradingScale for Healthy Adult and Adolescent Volunteers Enrolled in PreventiveVaccine Clinical Trials guidelines that were issued by the Food and DrugAdministration in September 2007. The DSMB reviewed laboratory and AEdata for the participants up to 8 weeks included in this report. Therewere protocol-specified safety stopping rules and adverse events ofspecial interest (AESIs). For the purpose of this report, clinical andlaboratory safety assessments up to 8 weeks post the first dose arepresented.

Protocol eligibility. Eligible participants must have met the followingcriteria: healthy adults aged between 18 and 50 years; able and willingto comply with all study procedures; Body Mass Index of 18-30 kg/m² atscreening; negative serological tests for Hepatitis B surface antigen,Hepatitis C antibody and Human Immunodeficiency Virus antibody;screening electrocardiogram (ECG) deemed by the Investigator as havingno clinically significant findings; use of medically effectivecontraception with a failure rate of <1% per year when used consistentlybe post-menopausal, or surgically sterile or have a partner who issterile. Key exclusion criteria included the following: individuals in acurrent occupation with high risk of exposure to SARS-CoV-2; previousknown exposure to SARS-CoV-2 or receipt of an investigational productfor the prevention or treatment of COVID-19; autoimmune orimmunosuppression as a result of underlying illness or treatment;hypersensitivity or severe allergic reactions to vaccines or drugs;medical conditions that increased risk for severe COVID-19; reportedsmoking, vaping, or active drug, alcohol or substance abuse ordependence; and fewer than two acceptable sites available forintradermal injection and electroporation.

Clinical Trial Population:

Healthy adult volunteers between the ages of 18-50 years, inclusive.

Inclusion Criteria:

-   -   a. Adults aged 18 to 50 years, inclusive;    -   b. Judged to be healthy by the Investigator on the basis of        medical history, physical examination and vital signs performed        at Screening;    -   c. Able and willing to comply with all study procedures;    -   d. Screening laboratory results within normal limits or deemed        not clinically significant by the Investigator;    -   e. Negative serological tests for Hepatitis B surface antigen        (HBsAg), Hepatitis C antibody and Human Immunodeficiency Virus        (HIV) antibody screening;    -   f. Screening electrocardiogram (ECG) deemed by the Investigator        as having no clinically significant findings (e.g.        Wolff-Parkinson-White syndrome);    -   g. Use of medically effective contraception with a failure rate        of <1% per year when used consistently and correctly from        screening until 3 months following last dose, be        post-menopausal, be surgically sterile or have a partner who is        sterile.

Exclusion Criteria:

-   -   a. Pregnant or breastfeeding, or intending to become pregnant or        father children within the projected duration of the trial        starting with the screening visit until 3 months following last        dose;    -   b. Is currently participating in or has participated in a study        with an investigational product within 30 days preceding Day 0;    -   c. Previous exposure to SARS-CoV-2 (laboratory testing at the        Investigator's discretion) or receipt of an investigational        vaccine product for prevention of COVID-19, MERS or SARS;    -   d. Current or history of the following medical conditions:        -   Respiratory diseases (e.g., asthma, chronic obstructive            pulmonary disease);        -   Hypertension, sitting systolic blood pressure >150 mm Hg or            a diastolic blood pressure >95 mm Hg;        -   Malignancy within 5 years of screening;        -   Cardiovascular diseases (e.g., myocardial infarction,            congestive heart failure, cardiomyopathy or clinically            significant arrhythmias);    -   e. Immunosuppression as a result of underlying illness or        treatment including:        -   Primary immunodeficiencies;        -   Long term use (≥7 days) of oral or parenteral            glucocorticoids;        -   Current or anticipated use of disease modifying doses of            anti-rheumatic drugs and biologic disease modifying drugs;        -   History of solid organ or bone marrow transplantation;        -   Prior history of other clinically significant            immunosuppressive or clinically diagnosed autoimmune            disease.    -   f. Fewer than two acceptable sites available for ID injection        and EP considering the deltoid and anterolateral quadriceps        muscles;    -   g. Any physical examination findings and/or history of any        illness that, in the opinion of the study investigator, might        confound the results of the study or pose an additional risk to        the patient by their participation in the study.

Immunogenicity Assessment Methods

Samples collected at screening, Week 0 (prior to dose) and at Weeks 6and 8 were analyzed. Peripheral Blood Mono-nuclear Cells (PBMCs) wereisolated from blood samples by a standard overlay on ficoll hypaquefollowed by centrifugation. Isolated cells were frozen in 10% DMSO and90% fetal calf serum. The frozen PBMCs were stored in liquid nitrogenfor subsequent analyses. Serum samples were stored at −80° C. until usedto measure binding and neutralizing antibody titers.

SARS-CoV-2 Wildtype Virus Neutralization Assays

SARS-CoV-2/Australia/VIC01/2020 isolate neutralization assays wereperformed at Public Health England (Porton Down, UK). Neutralizing virustiters were measured in serum samples that had been heat-inactivated at56° C. for 30 min. SARS-CoV-2 (Australia/VIC01/2020 isolate44) wasdiluted to a concentration of 933 pfu/m land mixed 50:50 in 1% FCS/MEMcontaining 25 mM HEPES buffer with doubling serum dilutions. After a 1 hincubation at 37° C., the virus-antibody mixture was transferred toconfluent monolayers of Vero E6 cells (ECACC 85020206; PHE, UK). Viruswas allowed to adsorb onto cells at 37° C. for a further hour in anincubator, and the cell monolayer was overlaid with MEM/4% FBS/1.5% CMC.After 5 days incubation at 37° C., the plates were fixed, stained, with0.2% crystal violet solution (Sigma) in 25% methanol (v/v). Plaques werecounted.

S1+S2 Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA plates were coated with 2.0 mg/mL recombinant SARS-CoV-2 S1+S2spike protein (Acro Biosystems; SPN-052H8) and incubated overnight at2-8° C. The S1+S2 contains amino acids residues Val 16-Pro 1213 of thefull length spike protein, GenBank #QHD43416.1. It contains twomutations to stabilize the protein to the trimeric pre-fusion state(R683A, R685A) and also contains a C-terminal 10×His tag (SEQ ID NO:24). The plates were then washed with PBS with 0.05% Tween-20 (Sigma;P3563) and blocked (Starting Block, Thermo Scientific; 37,538) for 1-3 hat room temperature. Samples were serially diluted using blocking bufferand were added in duplicate, along with prepared controls, to the washedand blocked assay plates. The samples were incubated on the blockedassay plates for one hour at room temperature. Following sample andcontrol incubation, the plates were washed and a 1/1000 preparation ofanti-human IgG HRP conjugate (BD Pharmingen; 555,788) in blocking bufferwas then added to each well and allowed to incubate for 1 h at roomtemperature. The plates were washed and TMB substrate (KPL; 5120-0077)was then added and allowed to incubate at room temperature forapproximately 10 min. TMB Stop Solution (KPL; 5150-0021) was next addedand the plates read at 450 nm and 650 nm on a Synergy HTX Micro-plateReader (BioTek). The magnitude of the assay response was expressed astiters which were defined as the greatest reciprocal dilution factor ofthe greatest dilution serial dilution at which the plate correctedoptical density is 3 SD above background a subject's corresponding Week0.

SARS-CoV-2 Spike ELISpot Assay

Peripheral mononuclear cells (PBMCs) pre- and post-vaccination werestimulated in vitro with 15-mer peptides (overlapping by 9 residues)spanning the full-length consensus spike protein sequence. Cells wereincubated overnight in an incubator with peptide pools at aconcentration of 5 mg per ml in a precoated ELISpot plate, (Mab-Tech,Human IFN-g ELISpot Plus). The next day, cells were washed off, and theplates were developed via a biotinylated anti-IFN-g detection antibodyfollowed by a streptavidin-enzyme conjugate resulting in visible spots.Each spot corresponds to an individual cytokine-secreting cell. Afterplates were developed, spots were scanned and quantified using the CTLS6 Micro Analyzer (CTL) with Immuno-Capture and ImmunoSpot software.Values are shown as the background-subtracted average of measuredtriplicates. The ELISpot assay qualification determined that 12 spotforming units was the lower limit of detection. Thus, anything abovethis cutoff is considered to be a signal of an antigen specific cellularresponse.

INO-4800 SARS-CoV-2 Spike Flow Cytometry Assay

PBMCs were also used for Intracellular Cytokine Staining (ICS) analysisusing flow cytometry. One million PMBCs in 200 mL complete RPMI mediawere stimulated for six hours (37° C., 5% CO₂) with DMSO (negativecontrol), PMA and Ionomycin (positive control, 100 ng/mL and 2 mg/mL,respectively), or with the indicated peptide pools (225 μg/mL). Afterone hour of stimulation, Brefeldin A and Monensin (BD GolgiStop andGolgiPlug, 0.001% and 0.0015%, respectively) were added to blocksecretion of expressed cytokines. After stimulation the cells were movedto 4° C. overnight. Next, cells were washed in PBS for live/deadstaining (Life Technologies Live/Dead aqua fixable viability dye), andthen resuspended in FACS buffer (0.5% BSA, 2 mM EDTA, 20 mM HEPES).Next, extracellular markers were stained, the cells were fixed andpermeabilized (eBioscience™ Foxp3Kit) and then stained for the indicatedcytokines (Table 9) using fluorescently conjugated antibodies. FIGS. 22Aand 22B show representative gating strategies for CD4+ and CD8+ T cellsas well as examples of positive expression of IFN

, TNFα, IL-2 and IL-4.

Statistical Analysis

No formal power analysis was applicable to this trial. Descriptivestatistics were used to summarize the safety end-points: proportionswith AEs, administration site reactions, and AESIs through 8 weeks.Descriptive statistics were also used to summarize the immunogenicityendpoints: median responses (with 95% confidence intervals) andpercentage of responders for cellular results, and geometric mean titers(with 95% confidence intervals) and percentage of responders for humoralresults. Post-hoc analyses of post-vaccination minus pre-vaccinationpaired differences in SARS-CoV-2 neutralization responses (on thenatural log-scale, with a paired t-test), ELISpot responses (withWilcoxon signed-rank tests), and Intracellular Flow Assay responses(with Wilcoxon signed-rank tests) were performed.

Results

Study Population Demographics

A total of 55 participants were screened and 40 participants wereenrolled into the initial two groups (FIG. 16 ). The median age was 34.5years (range 18 to 50 years). Participants were 55% (22/40) male (Table6). Most participants were white (82.5%, 33/40).

Vaccine Safety and Tolerability

A total of 39 of 40 (97.5%) participants completed both doses; oneparticipant in the 2.0 mg group discontinued trial participation priorto receiving the second dose due to lack of transportation to theclinical sites, and discontinuation was unrelated to the study or thedosing (FIG. 16 ). All 39 remaining subjects completed the visit 8 weekspost-dose 1. There was a total of 11 local and systemic adverse events(AEs) reported by 8 weeks post-dose 1; six of these were deemed relatedto vaccine (Table 10). All AEs were Grade 1 (mild) in severity. Five ofthe six related AEs were injection site reactions including injectionsite pain (3) and erythema (2). One Grade 1 systemic AE related to thevaccine was nausea. All related AEs occurred on the dosing day when thesubjects received the first or second vaccination. There were no febrilereactions and no antipyretic medicine was used post vaccination. Nosubject discontinued the trial due to an AE. No serious adverse events(SAEs) nor adverse events of special interest (AESIs) were reported.There were no abnormal laboratory values that were deemed clinicallysignificant by the Investigators throughout the initial 8-week follow-upperiod. There was no increase in the number of participants whoexperienced AEs related to the vaccine in the 2.0 mg group (10%, 2/20),compared to that in the 1.0 mg group (15%, 3/20) (FIGS. 19A and 19B). Inaddition, there was no increase in frequencies of AEs with the seconddose over the first dose in both dose groups.

TABLE 10 Number of Adverse Events classified by MedDRA ® System OrganClass, severity, and investigator assigned relationship to studyvaccination MedDRA ® Not System Organ related to Related to Total ClassSeverity vaccination vaccination number Any system Mild 5 6 11  OrganClass Moderate — — — Severe — — — Gastrointestinal Mild 1 1 2 DisordersModerate — — — Severe — — — General Mild — 5 5 Disorders and Moderate —— — Administration Severe — — — Site Conditions Injury, Mild 2 — 2Poisoning, and Moderate — — — Procedural Severe — — — ComplicationsNeoplasm Mild 1 — 1 Benign, Moderate — — — Malignant and Severe — — —Unspecified Nervous System Mild 1 — 1 Disorders Moderate — — — Severe —— —

Immunogenicity

Thirty-eight subjects were included in the immunogenicity analyses. Inaddition to one subject in the 2.0 mg group who discontinued prior tocompleting dosing, one subject in the 1.0 mg group was deemedseropositive at baseline and was excluded. Data for this subject can befound in Table 11.

TABLE 11 Immune Responses for subject who was Sero-positive atenrollment, INO-4800 1.0 mg Dose Group Output at Output at Immune AssayWeek 0 Week 6 Neutralization Week 6 785 1089 Reciprocal Titer RBDBinding Antibody 1 1 Week 6 Reciprocal Titer S1 + S2 Binding Antibody 114580 Week 6 Reciprocal Titer IFN-gamma ELISpot Week 55.6 27.8 6SFU/10{circumflex over ( )}6 PBMC

Humoral Immune Responses

Sera was tested for the ability to bind S1+S2 spike protein. 89%(17/19)of participants in the 1.0 mg group and 95% (18/19) of participants inthe 2.0 mg group had an increase in serum IgG binding titers to S1+S2spike protein when compared to their pre-vaccination timepoint (Week 0),with the responder GMT of 655.5 (95% CI:255.6, 1681.0) and 994.2 (95%CI: 395.3, 2500.3) in the 1.0 mg and 2.0 mg groups, respectively (FIG.17B, FIG. 20 and Table 13). Sera was also tested for the ability toneutralize live virus by live virus PRNTIC50 neutralization assay. Thegeometric mean fold-rise at Week 6 relative to baseline was 10.8 with a95% CI of (4.4, 27.0) and 11.5 with a 95% CI of (5.3, 24.9) in the 1.0mg and 2.0 mg groups, respectively. In each group, there was astatistically significant increase at Week 6 over baseline (P<0.0001paired t-test, post-hoc analysis), FIG. 17A. At Week 6, the percentageof responders were 78% (14/18) and 84% (16/19) in the 1.0 mg and 2.0 mggroups, respectively (FIG. 17A and Table 13), and the respondergeometric mean titer (GMT) were 102.3 (95% CI: 37.4, 280.3) and 63.5(95% CI: 39.6, 101.8) in the 1.0 mg and 2.0 mg groups, respectively.Overall seroconversion (defined as those participants who respond withneutralization and/or binding anti-bodies to S protein) at Week 6 in 1.0mg and 2.0 mg dose group were 95% (18/19) for each group (Table 13).

Enzyme-Linked Immunospot (ELISpot)

The percentage of responders at week 8 was 74% (14/19) in the 1.0 mgdose group, and 100% (19/19) in the 2.0 mg dose group. These data takenwith the seroconversion data result in a 100% (19/19) overall immuneresponse in each group (Table 13, FIGS. 18A and 21 ). The Median SFU per106 PBMC was 46 (95% CI: 21.1, 142.2) and 71 (95% CI: 32.2-194.4) forthe responders in 1.0 mg and 2.0 mg dose groups, respectively. Themedian change at week 8 relative to base-line was 22.3 (95% CI: 2.2,63.4) and 62.8 (95% CI: 22.2, 191.1) in the respective groups, and ineach group, there were statistically significant increases over baseline(P=0.001 and P<0.0001, respectively, Wilcoxon matched-pairs signed ranktest, post-hoc analysis), FIG. 18A. It is also interesting to note that3 convalescent samples (all 3 with symptoms but non-hospitalized),tested by the ELISpot assay showed lower T cell responses, with a medianof 33, than the 2.0 mg dose group at Week 8 (FIG. 20 ). As shown inFIGS. 18B and 18G, the 2.0 mg group's T cell responses were mapped to 5epitope pools. Encouragingly, T cell responses were seen in all regionsof the spike protein, with the dominant pool encompassing the ReceptorBinding Domain region, followed by pools covering the N Terminal Domain,as well as the Fusion Peptide, Heptad Repeat 1 and the Central Helix.

Intracellular Flow Assay

The contribution of CD4+ and CD8+T cells to the cellular immune responseagainst INO-4800 was assessed by intracellular cytokine staining (ICS).In the 2.0 mg dose group, the median change from baseline to Week 6 inCD8+T cells producing IFN-

, TNF-α and/or IL-2 (Any Response) was 0.11 with a 95% CI of (−0.02,0.23); the change was significantly increased (P=0.0181, Wilcoxonmatched-pairs signed rank test, post-hoc analysis). owing chiefly tosignificant increases in IFN-

as well as TNF-α production (FIG. 18C). Also in the 2.0 mg dose group,the median change from baseline to Week 6 in CD4+T cells producing TNF-αwas 0.02 with a 95% CI of (0.01 to 0.09); the change was alsosignificantly increased (P=0.0020, Wilcoxon matched-pairs signed ranktest, post-hoc analysis, FIG. 18C). The composition of CD4+ or CD8+Tcells producing any cytokine (IFN-

or TNF-α or IL-2 following vaccination) was also assessed for surfacemarkers CCR7 and CD45RA to characterize effector (CCR7−CD45RA+),effector memory (CCR7−CD45RA−), and central memory (CCR7+CD45RA−) cells(FIG. 18D). In both dose groups, CD8+T cells producing cytokine inresponse to stimulation with SARS-CoV-2spike peptides were generallybalanced across the three populations, whereas CD4+T cells werepredominantly of the central memory phenotype (FIG. 18D). CD4+ and CD8+Tcells following vaccination were further explored for their ability toproduce more than one cytokine at a time and were encouraged to notethat nearly half (41%) of the CD8+ T cells in the 2.0 mg dose group weredual producing IFN-

and TNF-α (FIG. 18E). CD8+T cells producing cytokine in the 1.0 mg dosegroup were primarily monofunctional IFN-

producing cells (57%). The CD4+T cell compartment was alsopolyfunctional in nature with 6% and 9%, in the 1.0 mg and 2.0 mg dosegroups, respectively, producing all 3 cytokines, IFN-

, TNF-α, and IL-2 (Table 12).Th2 responses were also measured byassessing IL-4 production, and no statistically significant increases(Wilcoxon matched-pairs signed rank test, post-hoc analysis) wereobserved in either group post vaccination (FIG. 18F).

INO-4800 was well tolerated with a frequency of product-related Grade 1AEs of 15% (3/20 subjects) and 10% (2/20 subjects) of the participantsin 1.0 mg and 2.0 mg dose group, respectively. Only Grade 1 AEs werenoted in the study, which compares favorably with existing licensedvaccines. The safety profile of a successful COVID-19 vaccine isimportant and supports broad development of INO-4800 in at-riskpopulations who are at more serious risk of complications fromSARS-CoV-2 infection, including the elderly and those withcomorbidities. INO-4800 also generated balanced humoral and cellularimmune responses with all 38 evaluable participants displaying either orboth antibody or T cell responses following two doses of INO-4800.Humoral responses measured by binding or neutralizing antibodies wereobserved in 95% (18/19) of the participants in each dose group. Theneutralizing antibodies, measured by live virus neutralization assay,were seen in 78% (14/18) and 84% (16/19) of participants, and thecorresponding GMTs were 102.3 [95% CI (37.4, 280.3)] and 63.5[95% CI(39.6, 101.8)] for the 1.0 mg and 2.0 mg dose groups, respectively. Therange overlaps that of the PRNT IC50 titers reported from convalescentpatients as well as the PRNT IC50 titers in NHPs which were protected ina SARS-CoV-2 challenge. Furthermore, there was a statisticallysignificant increase in titers. It is important to note that all but onevaccine recipient that did not develop neutralizing antibody titersresponded positively in the T cell ELISpot assay, suggesting that theimmune responses generated by the vaccine are registering differentiallyin these assays. Cellular immune responses were observed in 74% (14/19)and 100% (19/19) of 1.0 mg and 2.0 mg dose groups, respectively.Importantly, INO-4800 generated T cell responses that were more frequentand with higher responder median responses (46 [95% CI (21.1, 142.2)]vs. 71 [95% CI (32.2, 194.4)] SFU 106 PBMC) in the 1.0 mg and 2.0 mgdose groups respectively. These T cell responses in the 2.0 mg dosegroup were higher in magnitude than convalescent samples tested (FIG.18A). Furthermore, there was a statistically significant increase inSFU. In the flow cytometric assays, both the 1.0 mg and 2.0 mg DoseGroups showed increases in cytokine production from both the CD4+ andCD8+T cell compartments, especially in the 2.0 mg group. The 2.0 mggroup exhibited a number of statistically significant cytokine outputs,including IFN-

and TNF-α and “any cytokine” from the CD8+T cell compartment and TNF-αfrom the CD4+T cell compartment (FIG. 18C).Of considerable importance isthat CD8+T cell responses in the 2.0 mg dose group were dominated bycells expressing both IFN-

and TNF-α with or without IL-2 (FIG. 18E and Table 12). In total, thesecells amounted to nearly half of the total CD8+T cell response (42.7%,Table 12).

In this Phase 1 trial, INO-4800 vaccination led to substantial T cellresponses with increased Th1 phenotype, measured by both IFN-

ELISpot as well as multiparametric flow cytometry, as evidenced byincreased expression of Th1-type cytokines IFN-

, TNF-α, and IL-2 (FIG. 18C). Assessment of cellular responses inducedby INO-4800 displayed the presence of SARS-CoV-2 specific CD4+ and CD8+Tcells exhibiting hallmarks of differentiation into both central andeffector memory cells, suggesting that a persistent cellular responsehas been established (FIG. 18D). Importantly, this was accomplishedwhile minimizing induction of IL-4, a prototypical Th2 cytokine (FIG.18F), supporting that this vaccine has an immune phenotype, along withinduction of protection in preclinical models, which makes it unlikelyto be a risk for induction of enhanced disease.

TABLE 12 Flow Cytometry Polyfunctionality 1.0 mg Cohort 2.0 mg CohortCD4 CD8 CD4 CD8 Cytokine Cytokine Cytokine Cytokine Parameter FrequencyFrequency Frequency Frequency Output (%) (%) (%) (%) IFN-gamma 31.2 56.729.5 27.1 only TNF-alpha only 20.4 14 20.9 11.2 IL-2 only 22.3 16.5 20.116.5 IFN-gamma and 8.0 9.7 6.7 40.6 TNF-alpha only IFN-gamma and 2.1 0.90.6 1.3 IL-2 only IL-2 and 9.6 0.7 13.5 1.2 TNF-alpha only IFN-gamma and6.4 1.5 8.7 2.1 IL-2 and TNF-alpha Percents listed are the contributionsof each output to the total cytokine response

TABLE 13 1.0 mg Cohort 2.0 mg Cohort Overall Responder Responders^(‡)Overall Responder Responders^(‡) Immune Assay Value Value n (%) ValueValue n (%) Neutralization  44.4 102.3 14/18 (78%)  34.9  63.5 16/19(84%) Week 6 GMT [14.6, 134.8]  [37.4, 280.3] [15.8, 77.2]  [39.6,101.8] Reciprocal Titer (1, 11647) (13, 11647) (1, 652) (13652) [95% CI](Range) S1 + S2 Binding 331.2 655.5 17/19 (89%) 691.4 994.2 18/19 (95%)Antibody Week [91.2, 1203.2] [255.6, 168.1]  [217.5, 2197.2] [395.3,2500.3] 6 GMT (1, 14580) (20, 14580)  (1, 14580)  (20, 14580) ReciprocalTiter [95% CI] (Range) Total N/A N/A 18/19 (95%) N/A N/A 18/19 (95%)Seroconversion (Response in S1 + S2 or Neutralization) IFN-gamma 26.2SFU  45.6 14/19 (74%) ^(μ) 71 SFU 71 SFU 19/19 (100%) ^(μ) ELISpot Week[10.0-64.4] [21.1, 142.2] [32.2-194.4] [32.2-194.4] 8 Median SFU (1,374.4)  (16.7, 374.4)  (8.9, 615.6) (8.9, 615.6) per [95% CI] (Range)Overall N/A N/A 19/19 (100%) N/A N/A 19/19 (100%) Immune Response Rate(Seroconversion or ELISpot) 1.0 mg Cohort excludes one subject withbaseline positive NP ELISA ^(‡)Response criteria: Live Neutralization-Week 6 PRNT IC₅₀ ≥10, or ≥4 if binding ELISA activity is seen; S1 + S2Binding - Week 6 Value >1; RBD Binding -Week 6 value >1; ELISpot - Value≥12 SFU over Week 0 ^(μ) Responders generated using Week 6 or Week 8data

Expanded Phase I Study

Approximately 120 healthy volunteers will be evaluated across three (3)dose levels (Study Groups). A total of 40 subjects will be enrolled intoeach Study Group. Enrollment into each Study Group will be stratified byage; n=20 for 18-50 years, n=10 for 51-64 years, and n=10≥65 years(Table 14).

Subjects will be adults aged at least 18 years; judged to be healthy bythe Investigator on the basis of medical history, physical examinationand vital signs performed at Screening; able and willing to comply withall study procedures; screening laboratory results within normal limitsfor testing laboratory or deemed not clinically significant by theInvestigator; Body Mass Index of 18-30 kg/m², inclusive, at Screening;negative serological tests for Hepatitis B surface antigen (HBsAg),Hepatitis C antibody and Human Immunodeficiency Virus (HIV) antibody atscreening; screening ECG deemed by the Investigator as having noclinically significant findings (e.g. Wolff-Parkinson-White syndrome);and must meet one of the following criteria with respect to reproductivecapacity: women who are post-menopausal as defined by spontaneousamenorrhea for ≥12 months; surgically sterile or have a partner who issterile; use of medically effective contraception. Exclusion criteriaare as follows: pregnant or breastfeeding, or intending to becomepregnant or father children within the projected duration of the trialstarting with the screening visit until 3 months following last dose;positive serum pregnancy test during screening or positive urinepregnancy test prior to dosing; currently participating in or hasparticipated in a study with an investigational product within 30 dayspreceding Day 0; previous exposure to severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) or receipt of an investigational product forthe prevention or treatment of COVID-19, middle east respiratorysyndrome (MERS), or severe acute respiratory syndrome (SARS); in acurrent occupation with high risk of exposure to SARS-CoV-2 (e.g.,health care workers or emergency response personnel having directinteractions with or providing direct care to patients); current orhistory of respiratory disease, hypersensitivity or severe allergicreactions to vaccines or drugs, diagnosis of diabetes mellitus,hypertension, malignancy within 5 years of screening, or cardiovasculardisease; immunosuppression as a result of underlying illness ortreatment, including primary immunodeficiencies, long term use (>7 days)of oral or parenteral glucocorticoids, current or anticipated use ofdisease-modifying doses of anti-rheumatic drugs and biologicdisease-modifying drugs, history of solid organ or bone marrowtransplantation, and prior history of other clinically significantimmunosuppressive or clinically diagnosed autoimmune disease; fewer thantwo acceptable sites available for ID injection and EP considering thedeltoid and anterolateral quadriceps muscles; or reported smoking,vaping, or active drug, alcohol or substance abuse or dependence; or anyphysical examination findings and/or history of any illness that, in theopinion of the study investigator, might confound the results of thestudy or pose an additional risk to the patient by their participationin the study.

All subjects will receive dosing on Day 0 and Week 4 (Table 15).Subjects who consent to receive the booster dose (Table 16) will receivethe booster dose no earlier than Week 12 in their dosing schedule withthe same dose previously received for their two-dose regimen (Day 0 andWeek 4). Safety and immunogenicity will be evaluated at 2 weeksfollowing the booster dose.

TABLE 14 No. INO-4800 Number Number INO-4800 Injections/EP Dose perTotal Study Total Subjects Age Dosing Dose per per Dosing DosingINO-4800 Group Subjects by Age (years) Weeks injection Visit Visit Dose1 40  20* 18-50 0, 4 (±5 1.0 mg 1 1.0 mg 3.0 mg 10 51-64 days), 10 ≥65Optional Booster^(b) 2 40  20* 18-50 0, 4 (±5 1.0 mg  2^(a) 2.0 mg 6.0mg 10 51-64 days), 10 ≥65 Optional Booster^(b) 3 40 20 18-50 0, 4 (±50.5 mg 1 0.5 mg 1.5 mg 10 51-64 days), 10 ≥65 Optional Booster^(b) Total120 * Base Study (Others in expanded study) ^(a)INO-4800 will beinjected ID followed by EP in an acceptable location on two differentlimbs at each dosing visit ^(b)Optional booster dose delivered noearlier than Week 12 in their dosing schedule with the same dosepreviously received for their two-dose regimen.

Subjects not receiving an optional booster dose will be followed to theEnd of Study (EOS) visit at Week 52 will be the End of Study (EOS) visit(Table 15). For subjects receiving an optional booster dose, the 48 WeekPost-Booster Dose Visit will be the EOS visit (Table 16).

Primary Objectives:

-   -   Evaluate the tolerability and safety of INO-4800 administered by        ID injection followed by EP in healthy adult volunteers    -   Evaluate the cellular and humoral immune response to INO-4800        administered by ID injection followed by EP

Primary Safety Endpoints:

-   -   Incidence of adverse events by system organ class (SOC),        preferred term (PT), severity and relationship to        investigational product. Percentage of Participants with Adverse        Events (AEs) [Time Frame: Baseline up to Week 52 (if not        receiving an optional booster dose) or the 48 Week Post-Booster        Dose Visit (if receiving an optional booster dose)].    -   Administration (i.e., injection) site reactions (described by        frequency and severity). Percentage of Participants with        Administration (Injection) Site Reactions [Time Frame: Day 0 up        to Week 52 (if not receiving an optional booster dose) or the 48        Week Post-Booster Dose Visit (if receiving an optional booster        dose)].    -   Incidence of adverse events of special interest. Percentage of        Participants with Adverse Events of Special Interest (AESIs).        [Time Frame: Baseline up to Week 52 (if not receiving an        optional booster dose) or the 48 Week Post-Booster Dose Visit        (if receiving an optional booster dose)].

Primary Immunogenicity Endpoints:

-   -   SARS-CoV-2 Spike glycoprotein antigen-specific antibodies by        binding assays. Change from Baseline in SARS-CoV-2 Spike        Glycoprotein Antigen-Specific Binding Antibody Titers [Time        Frame: Baseline up to Week 52 (if not receiving an optional        booster dose) or the 48 Week Post-Booster Dose Visit (if        receiving an optional booster dose)].    -   Antigen-specific cellular immune response by IFN-gamma ELISpot        and/or flow cytometry assays. Change from Baseline in        Antigen-Specific Cellular Immune Response [Time Frame: Baseline        up to Week 52 (if not receiving an optional booster dose) or the        48 Week Post-Booster Dose Visit (if receiving an optional        booster dose)].

Exploratory Objectives:

-   -   Evaluate the expanded immunological profile by assessing both T        and B cell immune response    -   Evaluate the safety and immunogenicity of an optional booster        dose of INO-4800 administered by ID injection followed by EP        subsequent to a two-dose regimen Exploratory Endpoints:    -   Expanded immunological profile which may include (but not        limited to) additional assessment of T and B cell numbers,        neutralization response and T and B cell molecular changes by        measuring immunologic proteins and mRNA levels of genes of        interest at all weeks as determined by sample availability    -   Incidence of all adverse events subsequent to an optional        booster dose of INO-4800 administered by ID injection followed        by EP    -   SARS-CoV-2 Spike glycoprotein antigen-specific neutralizing and        binding antibodies subsequent to an optional booster dose of        INO-4800 administered by ID injection followed by EP    -   Antigen-specific cellular immune response by IFN-        ELISpot and/or flow cytometry subsequent to an optional booster        dose of INO-4800 administered by ID injection followed by EP

Safety Assessment:

Subjects will be followed for safety for the duration of the trialthrough EOS or the subject's last visit. Adverse events will becollected at every visit (including the Day 1 and 36 Week

Post-Booster Dose phone calls). Laboratory blood and urine samples willbe drawn according to the Schedule of Events (Table 15 and Table 16).

TABLE 15 NON-BOOSTER CLINICAL TRIAL SCHEDULE OF EVENTS Week 4 Day 0 Day1 Week 1 (±5 d) Week 6 Tests and assessments Screen^(a) Pre Post (+1 d)(±3 d) Pre Post (±5 d) Informed Consent X Inclusion/Exclusion Criteria XMedical History X X Demographics X Concomitant Medications X X X X XPhysical Exam^(b) X X X X X Vital Signs X X X X X Height and Weight XCBC with Differential X X X Chemistry^(c) X X X HIV, HBV, HCVSerology^(d) X SARS-CoV-2 Serology X 12-lead ECG X UrinalysisRoutine^(e) X X X Pregnancy Test^(f) X X X INO-4800 + EP^(g)  X^(h) X^(h) Download EP Data^(i) X X Adverse Events^(j) X X X X^(k) X X X XImmunology (Whole blood)^(l) X X X X Immunology (Serum)^(m) X X X X Week8 Week 12 Week 28 Week 40 Week 52 Tests and assessments (±5 d) (±5 d)(±5 d) (+5 d) (±5 d) Informed Consent Inclusion/Exclusion CriteriaMedical History Demographics Concomitant Medications X X X X X PhysicalExam^(b) X X X X X Vital Signs X X X X X Height and Weight CBC withDifferential X X X X X Chemistry^(c) X X X X X HIV, HBV, HCVSerology^(d) SARS-CoV-2 Serology 12-lead ECG Urinalysis Routine^(e) X XX X X Pregnancy Test^(f) INO-4800 + EP^(g) Download EP Data^(i) AdverseEvents^(j) X X X X X Immunology (Whole blood)^(l) X X X X X Immunology(Serum)^(m) X X X X X ^(a)Screening assessment occurs from −30 days to−1 day prior to Day 0. ^(b)Full physical examination at screening andWeek 52 (or any other study discontinuation visit) only. Targetedphysical exam at all other visits. ^(c)Includes Na, K, Cl, HCO3, Ca,PO4, glucose, BUN, Cr, AST, ALT and TBili. ^(d)HIV antibody or rapidtest, HBsAg, HCV antibody. ^(e)Dipstick for glucose, protein, andhematuria. Microscopic examination should be performed if dipstick isabnormal. ^(f)Serum pregnancy test at screening. Urine pregnancy test atother visits. ^(g)All doses delivered via intradermal injection followedby EP. ^(h)For Study Groups Groups 1 and 3, one injection in skinpreferably over deltiod muscle at Day 0 and Week 4. For Study Group 2,two injections in skin with each injection over a different deltoid orlateral quadriceps; preferably over the deltoid muscles, at Day 0 andWeek 4. ^(i)Following administration of INO-4800 + EP, EP data will bedownloaded from the CELLECTRA ® 2000 device and provided to Inovio.^(j)Includes AEs from the time of consent and all injection sitereactions that qualify as an AE. ^(k)Follow-up phone call to collectAEs. ^(l)4 × 8.5 mL (34 mL) whole blood in 10 mL Acid Citrate Dextrose(ACD, Yellow top) tubes per time point. Note: Collect a total of 68 mLwhole blood prior to 1st dose (screening and prior to Day 0 dosing).^(m)1 × 8 mL blood in 10 mL red top serum collection tube per timepoint. Note: Collect four aliquots of 1 mL each (total 4 mL) serum ateach time point prior to 1st dose (Screening and prior to Day 0 dosing).

TABLE 16 Booster Clinical Trial Schedule of Events 2 Week 12 Week 24Week 36 Week 48 Week Post- Booster Dose Post-Booster Post-BoosterPost-Booster Post-Booster Booster Dose Visit Dose Visit Dose Visit DoseVisit Dose Phone Visit Tests and assessments Pre Post (±5 d) (±5 d) (±5d) Call (+5 d) (±5 d) Concomitant Medications X X X X X PhysicalExam^(a) X X X X X Vital Signs X X X X X CBC with Differential X X X X XChemistry^(b) X X X X X Urinalysis Routine^(c) X X X X X PregnancyTest^(d) X INO-4800 + EP^(e)  X^(f) Download EP Data^(g) X AdverseEvents^(h) X X X X X X^(i) X Immunology (Whole blood)^(j) X X X X XImmunology (Serum)^(k) X X X X X ^(a)Full physical examination at the 48Week Post-Booster Dose Visit (or any other study discontinuation visit)only. Targeted physical exam at all other visits. ^(b)Includes Na, K,Cl, HCO₃, Ca, PO₄, glucose, BUN, Cr, AST, ALT and TBili. ^(c)Dipstickfor glucose, protein, and hematuria. Microscopic examination should beperformed if dipstick is abnormal. ^(d)Urine pregnancy test must benegative prior to receiving booster dose. ^(e)All doses delivered viaintradermal injection followed by EP. ^(f)For Study Groups 1 and 3, oneinjection in skin preferably over deltiod muscle (or alternatively,lateral quadriceps) at the Booster Dose Visit. For Study Group 2, twoinjections in skin with each injection over a different deltoid orlateral quadriceps; preferably over the deltoid muscles, at the BoosterDose Visit. ^(g)Following administration of INO-4800 + EP, EP data willbe downloaded from the CELLECTRA ® 2000 device and provided to Inovio.^(h)Includes AEs from the time of consent and all injection sitereactions that qualify as an AE. ^(i)Follow-up phone call to collectAEs. ^(j)4 × 8.5 mL (34 mL) whole blood in 10 mL Acid Citrate Dextrose(ACD, Yellow top) tubes per time point. ^(k)1 × 8 mL blood in 10 mL redtop serum collection tube per time point.

Immunogenicity Assessment:

Immunology blood samples will be collected according to the Schedule ofEvents (Table 15 and Table 16). Determination of analysis of collectedsamples for immunological endpoints will be determined on an ongoingbasis throughout the study.

INO-4800 delivered ID followed by EP using CELLECTRA® 2000 in healthyvolunteers is expected to be well tolerated, exhibit an acceptablesafety profile, and result in generation of immune responses toSARS-CoV-2 Spike glycoprotein.

Example 7 Phase 2/3 Randomized, Blinded, Placebo-Controlled Trial toEvaluate the Safety, Immunogenicity, and Efficacy of INO-4800, aProphylactic Vaccine Against COVID-19 Disease, AdministeredIntradermally Followed by Electroporation (EP) in Healthy SeronegativeAdults at High Risk of SARS-CoV-2 Exposure

This is a Phase 2/3, randomized, placebo-controlled, multi-center trialto evaluate the safety, immunogenicity and efficacy of INO-4800administered by intradermal (ID) injection followed by electroporation(EP) using CELLECTRA® 2000 device to prevent COVID-19 disease inparticipants at high risk of exposure to SARS-CoV-2. The Phase 2 segmentwill evaluate immunogenicity and safety in approximately 400participants at two dose levels across three age groups. Safety andimmunogenicity information from the Phase 2 segment will be used todetermine the dose level for the Phase 3 efficacy segment of the studyinvolving approximately 6178 participants.

TABLE 17 Arm Intervention/treatment Experimental Phase 2: INO-4800 DoseDrug: INO-4800 Group 1 INO-4800 will be administered Participants willreceive one ID on Day 0 and Day 28. intradermal (ID) injection of 1.0Device: CELLECTRA ® 2000 milligram (mg) of INO-4800 EP using theCELLECTRA ® followed by electroporation (EP) 2000 device will beadministered using the CELLECTRA® 2000 following ID delivery of INO-device on Day 0 and Day 28. 4800 on Day 0 and Day 28. Experimental:Phase 2: INO-4800 Dose Drug: INO-4800 Group 2 INO-4800 will beadministered Participants will receive two ID ID on Day 0 and Day 28.injections of 1.0 mg (total 2.0 mg per Device: CELLECTRA ® 2000 dosingvisit) of INO-4800 followed by EP using the CELLECTRA ® EP using theCELLECTRA ® 2000 2000 device will be administered device on Day 0 andDay 28. following ID delivery of INO- 4800 on Day 0 and Day 28. PlaceboComparator: Phase 2: Placebo Drug: Placebo Dose Group 1 Sterile salinesodium citrate Participants will receive one ID (SSC) buffer (SSC-0001)will be injection of placebo followed by EP administered ID on Day 0 andusing the CELLECTRA ® 2000 Day 28. device on Day 0 and Day 28. OtherNames: SSC-0001 Device: CELLECTRA ® 2000 EP using the CELLECTRA ® 2000device will be administered following ID delivery of sterile salinesodium citrate (SSC) buffer (SSC-0001) on Day 0 and Day 28. PlaceboComparator: Phase 2: Placebo Drug: Placebo Dose Group 2 Sterile salinesodium citrate Participants will receive two ID (SSC) buffer (SSC-0001)will be injections of placebo followed by EP administered ID on Day 0and using the CELLECTRA ® 2000 Day 28. device on Day 0 and Day 28. OtherNames: SSC-0001 Device: CELLECTRA ® 2000 EP using the CELLECTRA ® 2000device will be administered following ID delivery of sterile salinesodium citrate (SSC) buffer (SSC-0001) on Day 0 and Day 28.Experimental: Phase 3: INO-4800 Drug: INO-4800 Optimum Dose INO-4800will be administered Participants will receive either one or ID on Day 0and Day 28. two 1.0 mg ID injections of INO-4800 Device: CELLECTRA ®2000 based on results from Phase 2 EP using the CELLECTRA ® segment,followed by EP using the 2000 device will be administered CELLECTRA ®2000 device on following ID delivery of INO- Day 0 and Day 28. 4800 onDay 0 and Day 28. Placebo Comparator: Phase 3: Placebo Drug: PlaceboOptimum Dose Sterile saline sodium citrate Participants will receiveeither one or (SSC) buffer (SSC-0001) will be two ID injections ofplacebo based on administered ID on Day 0 and results from Phase 2segment, Day 28. followed by EP using the Other Names: CELLECTRA ® 2000device on SSC-0001 Day 0 and Day 28. Device: CELLECTRA ® 2000 EP usingthe CELLECTRA ® 2000 device will be administered following ID deliveryof sterile saline sodium citrate (SSC) buffer (SSC-0001) on Day 0 andDay 28.

Primary Outcome Measure:

1. Phase 2: Change From Baseline in Antigen-specific Cellular ImmuneResponse Measured by Interferon-gamma (IFN-γ) Enzyme-linked Immunospot(ELISpot) Assay [Time Frame: Baseline up to Day 393]

2. Phase 2: Change From Baseline in Neutralizing Antibody ResponseMeasured by a Pseudovirus-based Neutralization Assay [Time Frame:Baseline up to Day 393]

3. Percentage of Participants With Virologically Confirmed COVID-19Disease [Time Frame: From 14 days after completion of the 2-dose regimenup to 12 months post-dose 2 (i.e. Day 42 up to Day 393)]

Secondary Outcome Measures:

1. Phase 2 and 3: Percentage of Participants with Unsolicited andSolicited Injection Site Reactions [Time Frame: From time of consent upto 28 days post-dose 2 (up to Day 56)]

2. Phase 2 and 3: Percentage of Participants with Solicited andUnsolicited Systemic Adverse Events (AEs) [Time Frame: From time ofconsent up to 28 days post-dose 2 (up to Day 56)]

3. Phase 2 and 3: Percentage of Participants with Serious Adverse Events(SAEs) [Time Frame: Baseline up to Day 393]

4. Phase 2 and 3: Percentage of Participants with Adverse Events ofSpecial Interest (AESIs) [Time Frame: Baseline up to Day 393]

5. Phase 3: Percentage of Participants With Death from All Causes [TimeFrame: Baseline up to Day 393]

6. Phase 3: Percentage of Participants With Non-Severe COVID-19 Disease[Time Frame: From 14 days after completion of the 2-dose regimen up to12 months post-dose 2 (i.e. Day 42 up to Day 393)]

7. Phase 3: Percentage of Participants With Severe COVID-19 Disease[Time Frame: From 14 days after completion of the 2-dose regimen up to12 months post-dose 2 (i.e. Day 42 up to Day 393)]

8. Phase 3: Percentage of Participant With Death from COVID-19 Disease[Time Frame: From 14 days after completion of the 2-dose regimen up to12 months post-dose 2 (i.e. Day 42 up to Day 393)]

9. Phase 3: Percentage of Participants With Virologically-ConfirmedSARS-CoV-2 Infections [Time Frame: From 14 days after completion of the2-dose regimen up to 12 months post-dose 2 (i.e. Day 42 up to Day 393)]

10. Phase 3: Days to Symptom Resolution in Participants With COVID-19Disease [Time Frame: From 14 days after completion of the 2-dose regimenup to 12 months post-dose 2 (i.e. Day 42 up to Day 393)]

11. Phase 3: Change From Baseline in Antigen-specific Cellular ImmuneResponse Measured by IFN-gamma ELISpot Assay [Time Frame: Baseline up toDay 393]

12. Phase 3: Change From Baseline in Neutralizing Antibody ResponseMeasured by a Pseudovirus-based Neutralization Assay [Time Frame:Baseline up to Day 393]

Eligibility Criteria

Ages Eligible for Study: 18 Years and older

Sexes Eligible for Study: All

Gender Based: No

Accepts Healthy Volunteers: Yes

Key Inclusion Criteria:

-   -   Working or residing in an environment with high risk of exposure        to SARS-CoV-2 for whom exposure may be relatively prolonged or        for whom personal protective equipment (PPE) may be        inconsistently used, especially in confined settings    -   Screening laboratory results within normal limits for testing        laboratory or are deemed not clinically significant by the        Investigator.    -   Be post-menopausal or be surgically sterile or have a partner        who is sterile or use medically effective contraception with a        failure rate of <1% per year when used consistently and        correctly from screening until 3 months following last dose.

Key Exclusion Criteria:

-   -   Acute febrile illness with temperature >100.4° F. (38.0° C.) or        acute onset of upper or lower respiratory tract symptoms (e.g.,        cough, shortness of breath, sore throat).    -   Positive serologic or molecular (Reverse transcription        polymerase chain reaction [RT-PCR]) test for SARS-CoV-2 at        Screening    -   Pregnant or breastfeeding or intending to become pregnant or        intending to father children within the projected duration of        the trial starting from the screening visit until 3 months        following the last dose.    -   Known history of uncontrolled HIV based on a CD4 count less than        200 cells per cubic millimeter (/mm{circumflex over ( )}3) or a        detectable viral load within the past 3 months.    -   Is currently participating or has participated in a study with        an investigational product within 30 days preceding Day 0.    -   Previous receipt of an investigational vaccine for prevention or        treatment of COVID-19, middle east respiratory syndrome (MERS),        or severe acute respiratory syndrome (SARS) (documented receipt        of placebo in previous trial would be permissible for trial        eligibility).    -   Respiratory diseases (e.g., asthma, chronic obstructive        pulmonary disease) requiring significant changes in therapy or        hospitalization for worsening disease during the 6 weeks prior        to enrollment.    -   Immunosuppression as a result of underlying illness or treatment    -   Lack of acceptable sites available for ID injection and EP    -   Blood donation or transfusion within 1 month prior to Day 0.    -   Reported alcohol or substance abuse or dependence, or illicit        drug use (excluding marijuana use).    -   Any illness or condition that in the opinion of the investigator        may affect the safety of the participant or the evaluation of        any study endpoint.

Example 8 One or Two Dose Regimen of the SARS-CoV-2 DNA Vaccine INO-4800Protects Against Respiratory Tract Disease Burden in Nonhuman Primate(NHP) Challenge Model

The safety, immunogenicity and efficacy of the intradermal delivery ofINO-4800, a synthetic DNA vaccine candidate encoding a SARS-CoV-2 spikeantigen, was evaluated in the rhesus macaque model. Single and two dosevaccination regimens were evaluated. Vaccination induced both bindingand neutralizing antibodies, along with IFN-γ-producing T cells againstSARS-CoV-2. A high dose of SARS-CoV-2 Victoria01 strain (5×10{circumflexover ( )}6 pfu) was used to specifically assess the impact of INO-4800vaccination on lung disease burden to provide both vaccine safety andefficacy data. A broad range of lower respiratory tract diseaseparameters were measured by applying histopathology, lung diseasescoring metric system, in situ hybridization, viral RNA RT-PCR andcomputed tomography (CT) scans to provide an understanding of the impactof vaccine induced immunity on protective efficacy and potential vaccineenhanced disease (VED).

This example describes the immunogenicity, efficacy and safetyassessment of the SARS-CoV-2 DNA vaccine INO-4800 in a stringent highdose nonhuman primate challenge model. Intradermal delivery of 1 mg ofINO-4800 to rhesus macaques induces humoral and T cell responses againstthe SARS-CoV-2 spike antigen in both a 2-dose regimen and a suboptimal 1dose regimen. Throughout the study no overt clinical events wererecorded in the animals. After a high dose SARS-CoV-2 challenge, areduction in viral loads was observed and lung disease burden in boththe 1 and 2 dose vaccine groups supporting the efficacy of INO-4800.Importantly, vaccine enhanced disease (VED) was not observed, even withthe 1 dose group.

Methods

Vaccine. The optimized DNA sequence encoding SARS-CoV-2 IgELS-spike wascreated using Inovio's proprietary in silico Gene Optimization Algorithmto enhance expression and immunogenicity. The optimized DNA sequence wassynthesized, digested with BamHI and XhoI, and cloned into theexpression vector pGX0001 under the control of the human cytomegalovirusimmediate-early promoter and a bovine growth hormone polyadenylationsignal.

Animals. Eighteen rhesus macaques of Indian origin (Macaca mulatta) wereused in this study. Study groups comprised three males and three femalesof each species and all were adults aged between 2.5 and 3.5 years ofage and weighing >4 Kg at time of challenge. Prior to the start of theexperiment, socially compatible animals were randomly assigned tochallenge groups, to minimize bias. Animals were housed in compatiblesocial groups, in cages in accordance with the UK Home Office Code ofPractice for the Housing and Care of Animals Bred, Supplied or Used forScientific Procedures (2014) and National Committee for Refinement,Reduction and Replacement (NC3Rs) Guidelines on Primate Accommodation,Care and Use, August 2006. Housing prior and for the duration ofchallenge is described in [Salguero, F. J., et al., Comparison of Rhesusand Cynomolgus macaques as an authentic model for COVID-19. bioRxiv,2020: p. 2020.09.17.3010931. All experimental work was conducted underthe authority of a UK Home Office approved project license (PDC57C033)that had been subject to local ethical review at PHE Porton Down by theAnimal Welfare and Ethical Review Body (AWERB) and approved as requiredby the Home Office Animals (Scientific Procedures) Act 1986. Animalswere sedated by intramuscular (IM) injection with ketamine hydrochloride(Ketaset, 100 mg/ml, Fort Dodge Animal Health Ltd, Southampton, UK; 10mg/kg) for procedures requiring removal from their housing. None of theanimals had been used previously for experimental procedures.

Vaccine administration. Animals received 1 mg of SARS-CoV-2 DNA vaccine,INO-4800, by intradermal injection at day 28 only (1 dose group) or 0and 28 (2 dose group) followed by an EP treatment using the CELLECTRA2000® Adaptive Constant Current Electroporation Device with a 3P array(Inovio Pharmaceuticals).

Serum and heparinised whole blood were collected whilst animals weresedated at bi-weekly intervals during the vaccination phase. Nasal andthroat swabs were also collected on the day of challenge on D56. Afterchallenge, nasal swabs, throat swabs and serum were collected at 1, 3, 5dpc and at cull (6, 7 or 8 dpc—staggered due to the high level of laborinvolved in procedures), with heparinised whole blood collected at 3 dpcand at cull. Nasal and throat swabs were obtained as described[Salguero, F. J., et al., Comparison of Rhesus and Cynomolgus macaquesas an authentic model for COVID-19. bioRxiv, 2020: p. 2020.09.17.3010931

Clinical observations. Animals were monitored multiple times per day forbehavioral and clinical changes. Behavior was evaluated forcontra-indicators including depression, withdrawal from the group,aggression, changes in feeding patterns, breathing pattern, respirationrate and cough. Animals were observed and scored as follows for activityand health throughout the study. Key: Activity Level: A0=Active & Alert;A1=Only active when stimulated by operator; A2=Inactive even whenstimulated/Immobile; H=Healthy; S=Sneeze, C=Cough, Nd=Nasal Discharge,Od=Ocular Discharge, Rn=Respiratory Noises, Lb=Laboured breathing,L=Lethargy, Di=Diarrhoea, Ax=Loss of Appetite, Dx=Dehydration,RD=Respiratory Distress. Animal body weight, temperature and haemoglobinlevels were measured and recorded throughout the study.

Viruses and Cells

SARS-CoV-2 Victoria/01/2020 [Caly, L., et al., Isolation and rapidsharing of the 2019 novel coronavirus (SARS-CoV-2) from the firstpatient diagnosed with COVID-19 in Australia. Med J Aust, 2020. 212(10):p. 459-462] was generously provided by The Doherty Institute, Melbourne,Australia at P1 after primary growth in Vero/hSLAM cells andsubsequently passaged twice at PHE Porton Down in Vero/hSLAM cells[ECACC 04091501]. Infection of cells was with 0.0005 MOI of virus andharvested at day 4 by dissociation of the remaining attached cells bygentle rocking with sterile 5 mm borosilicate beads followed byclarification by centrifugation at 1,000×g for 10 mins. Whole genomesequencing was performed, on the P3 challenge stock, using both Nanoporeand Illumina as described in Lewandowski, K., et al., MetagenomicNanopore Sequencing of Influenza Virus Direct from Clinical RespiratorySamples. J Clin Microbiol, 2019. 58(1). Virus titer of the challengestocks was determined by plaque assay on Vero/E6 cells [ECACC 85020206].Cell lines were obtained from the European Collection of AuthenticatedCell Cultures (ECACC) PHE, Porton Down, UK. Cell cultures weremaintained at 37° C. in Minimum essential medium (MEM) (LifeTechnologies, California, USA) supplemented with 10% fetal bovine serum(FBS) (Sigma, Dorset, UK) and 25 mM HEPES (Life Technologies,California, USA). In addition, Vero/hSLAM cultures were supplementedwith 0.4 mg/ml of geneticin (Invitrogen) to maintain the expressionplasmid. Challenge substance dilutions were conducted in phosphatebuffer saline (PBS). Inoculum (5×10⁶ PFU) was delivered by intratrachealroute (2 ml) and intranasal instillation (1.0 ml total, 0.5 ml pernostril).

Clinical Signs and In-Life Imaging by Computerized Tomography

CT scans were performed two weeks before and five days after challengewith SARS-CoV2. CT imaging was performed on sedated animals using a 16slice Lightspeed CT scanner (General Electric Healthcare, Milwaukee, WI,USA) in both the prone and supine position and scans evaluated by amedical radiologist expert in respiratory diseases (as describedpreviously [Salguero, F. J., et al., Comparison of Rhesus and Cynomolgusmacaques as an authentic model for COVID-19. 2020: p.2020.09.17.301093.]). To provide the power to discriminate differencesbetween individual NHP's with low disease volume (i.e. <25% lunginvolvement), a refined score system was designed in which scores wereattributed for possession of abnormal features characteristic of COVIDin human patients (COVID pattern score) and for the distribution offeatures through the lung (Zone score). The COVID pattern score wascalculated as sum of scores assigned for the number of nodulesidentified, and the possession and extent of GGO and consolidationaccording to the following system: Nodule(s): Score 1 for 1, 2 for 2 or3, 3 for 4 or more; GGO: each affected area was attributed with a scoreaccording to the following: Score 1 if area measured <1 cm, 2 if 1 to 2cm, 3 if 2 −3 cm, 4 if >3 cm and scores for each area of GGO were summedto provide a total GGO score; Consolidation: each affected area wasattributed with a score according to the following: 1 if area measured<1 cm, 2 if 1 to 2 cm, 3 if 2-3 cm, 4 if >3 cm. Scores for each area ofconsolidation are summed to provide a total consolidation score. Toaccount for estimated additional disease impact on the host ofconsolidation compared to GGO, the score system was weighted by doublingthe score assigned for consolidation. To determine the zone score, thelung was divided into 12 zones and each side of the lung divided (fromtop to bottom) into three zones: the upper zone (above the carina), themiddle zone (from the carina to the inferior pulmonary vein), and thelower zone (below the inferior pulmonary vein). Each zone was furtherdivided into two areas: the anterior area (the area before the verticalline of the midpoint of the diaphragm in the sagittal position) and theposterior area (the area after the vertical line of the mid-point of thediaphragm in the sagittal position). This results in 12 zones in totalwhere a score of one is attributed to each zone containing structuralchanges. The COVID pattern score and the zone are summed to provide theTotal CT score.

Post-mortem examination and histopathology. Animals were euthanized at 3different time-points, in groups of six (including one animal from eachspecies and sex) at 6, 7 and 8 dpc. The bronchial alveolar lavage fluid(BAL) was collected at necropsy from the right lung. The left lung wasdissected prior to BAL collection and used for subsequent histopathologyand virology procedures. At necropsy nasal and throat swabs, heparinisedwhole blood and serum were taken alongside tissue samples forhistopathology. Samples from the left cranial and left caudal lung lobetogether with spleen, kidney, liver, mediastinal and axillary lymphnodes, small intestine (duodenum), large intestine (colon), trachea,larynx inoculation site and draining lymph node, were fixed by immersionin 10% neutral-buffered formalin and processed routinely into paraffinwax. Four μm sections were cut and stained with hematoxylin and eosin(H&E) and examined microscopically. A lung histopathology scoring system[Salguero, F. J., et al., Comparison of Rhesus and Cynomolgus macaquesas an authentic model for COVID-19. bioRxiv, 2020: p. 2020.09.17.301093]was used to evaluate lesions affecting the airways and the parenchyma.Three tissue sections from each left lung lobe were used to evaluate thelung histopathology. In addition, samples were stained using theRNAscope technique to identify the SARS-CoV-2 virus RNA in lung tissuesections. Briefly, tissues were pre-treated with hydrogen peroxide for10 mins (RT), target retrieval for 15 mins (98-102° C.) and proteaseplus for 30 mins (40° C.) (Advanced Cell Diagnostics). A V-nCoV2019-Sprobe (SARS-CoV-2 Spike gene specific) was incubated on the tissues fortwo hours at 40° C. In addition, samples were stained using the RNAscopetechnique to identify the SARS-CoV-2 virus RNA. Amplification of thesignal was carried out following the RNAscope protocol using theRNAscope 2.5 HD Detection kit—Red (Advanced Cell Diagnostics,Biotechne). All H&E and ISH stained slides were digitally scanned usinga Panoramic 3D-Histech scanner and viewed using CaseViewer v2.4software. The presence of viral RNA by ISH was evaluated using the wholelung tissue section slides. Digital image analysis was performed inRNAscope labelled slides to ascertain the percentage of stained cellswithin the lesions, by using the Nikon-NIS-Ar software package.

Viral load quantification by RT-qPCR. RNA was isolated from nasal swabsand throat swabs. Samples were inactivated in AVL (Qiagen) and ethanol.Downstream extraction was then performed using the BioSprint™96One-For-All vet kit (Indical) and Kingfisher Flex platform as permanufacturer's instructions. Tissues were homogenized in Buffer RLT+betamercaptoethanol (Qiagen). Tissue homogenate was then centrifugedthrough a QIAshredder homogenizer (Qiagen) and supplemented with ethanolas per manufacturer's instructions. Downstream extraction from tissuesamples was then performed using the BioSprint™96 One-For-All vet kit(Indical) and Kingfisher Flex platform as per manufacturer'sinstructions.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)targeting a region of the SARS-CoV-2 nucleocapsid (N) gene was used todetermine viral loads and was performed using TaqPath™ 1-Step RT-qPCRMaster Mix, CG (Applied Biosystems™) 2019-nCoV CDC RUO Kit (IntegratedDNA Technologies) and QuantStudio™ 7 Flex Real-Time PCR System.Sequences of the N1 primers and probe were: 2019-nCoV_N1-forward, 5′GACCCCAAAATCAGCGAAAT 3′ (SEQ ID NO: 18); 2019-nCoV_N1-reverse, 5′TCTGGTTACTGCCAGTTGAATCTG 3′(SEQ ID NO: 19); 2019-nCoV N1-probe, 5′FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1 3′(SEQ ID NO: 20). The cyclingconditions were: 25° C. for 2 minutes, 50° C. for 15 minutes, 95° C. for2 minutes, followed by 45 cycles of 95° C. for 3 seconds, 55° C. for 30seconds. The quantification standard was in vitro transcribed RNA of theSARS-CoV-2 N ORF (accession number NC_045512.2) with quantificationbetween 1 and 6 log copies/μl. Positive swab and fluid samples detectedbelow the limit of quantification (LoQ) of 4.11 log copies/ml, wereassigned the value of 5 copies/μl, this equates to 3.81 log copies/ml,whilst undetected samples were assigned the value of <2.3 copies/μl,equivalent to the assay's lower limit of detection (LoD) which equatesto 3.47 log copies/ml. Positive tissue samples detected below the limitof quantification (LoQ) of 4.76 log copies/ml were assigned the value of5 copies/μl, this equates to 4.46 log copies/g, whilst undetectedsamples were assigned the value of <2.3 copies/μl, equivalent to theassay's lower limit of detection (LoD) which equates to 4.76 logcopies/g.

Subgenomic RT-qPCR was performed on the QuantStudio™ 7 Flex Real-TimePCR System using TaqMan™ Fast Virus 1-Step Master Mix (Thermo FisherScientific) and oligonucleotides as specified by Wölfel, et al.Virological assessment of hospitalized patients with COVID-2019. Nature581, 465-469 (2020), with forward primer, probe and reverse primer at afinal concentration of 250 nM, 125 nM and 500 nM respectively. Sequencesof the sgE primers and probe were:

2019-nCoV_sgE-forward, (SEQ ID NO: 21) 5′ CGATCTCTTGTAGATCTGTTCTC 3′;2019-nCoV_sgE-reverse, (SEQ ID NO: 22) 5′ ATATTGCAGCAGTACGCACACA 3′;2019-nCoV_sgE-probe, (SEQ ID NO: 23)5′ FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1 3′.

Cycling conditions were 50° C. for 10 minutes, 95° C. for 2 minutes,followed by 45 cycles of 95° C. for 10 seconds and 60° C. for 30seconds. RT-qPCR amplicons were quantified against an in vitrotranscribed RNA standard of the full length SARS-CoV-2 E ORF (accessionnumber NC_045512.2) preceded by the UTR leader sequence and putative Egene transcription regulatory sequence described by Wolfel et al[Wölfel, R., Corman, V. M., Guggemos, W. et al. Virological assessmentof hospitalized patients with COVID-2019. Nature 581, 465-469 (2020).].Positive samples detected below the lower limit of quantification (LLOQ)were assigned the value of 5 copies/μl, whilst undetected samples wereassigned the value of ≤0.9 copies/μl, equivalent to the assays lowerlimit of detection (LLOD). For nasal swab, throat swab and BAL samplesextracted samples this equates to an LLOQ of 4.11 log copies/mL and LLODof 3.06 log copies/mL. For tissue samples this equates to an LLOQ of4.76 log copies/g and LLOD of 3.71 log copies/g.

Plaque reduction neutralization test. Neutralizing virus titers weremeasured in heat-inactivated (56° C. for 30 minutes) serum samples.SARS-CoV-2 was diluted to a concentration of 1.4×10³ pfu/ml (70 pfu/50μl) and mixed 50:50 in 1% FCS/MEM with doubling serum dilutions from1:10 to 1:320 in a 96-well V-bottomed plate. The plate was incubated at37° C. in a humidified box for one hour to allow the antibody in theserum samples to neutralize the virus. The neutralized virus wastransferred into the wells of a washed plaque assay 24-well plate (seeplaque assay method), allowed to adsorb at 37° C. for a further hour,and overlaid with plaque assay overlay media. After five days incubationat 37° C. in a humified box, the plates were fixed, stained and plaquescounted.

Antigen Binding ELISA. Recombinant SARS-CoV-2 Spike- and RBD-specificIgG responses were determined by ELISA. A full-length trimeric andstabilized version of the SARS-CoV-2 Spike protein was supplied by LakePharma (#46328). Recombinant SARS-CoV-2 Receptor-Binding-Domain(319-541) Myc-His was developed and kindly provided by MassBiologics.High-binding 96-well plates (Nunc Maxisorp, 442404) were coated with 50μl per well of 2 μg/ml Spike trimer (S1+S2) or RBD in 1×PBS (Gibco) andincubated overnight at 4° C. The ELISA plates were washed and blockedwith 5% Fetal Bovine Serum (FBS, Sigma, F9665) in 1×PBS/0.1% Tween 20for 1 hour at room temperature. Serum collected from animals aftervaccination had a starting dilution of 1/50 followed by 8 two-foldserial dilutions. Post-challenge samples were inactivated in 0.5% tritonand had a starting dilution of 1/100 followed by 8 three-fold serialdilutions. Serial dilutions were performed in 10% FBS in 1×PBS/0.1%Tween 20. After washing the plates, 50 μl/well of each serum dilutionwas added to the antigen-coated plate in duplicate and incubated for 2hours at room temperature. Following washing, anti-monkey IgG conjugatedto HRP (Invitrogen, PA1-84631) was diluted (1:10,000) in 10% FBS in1×PBS/0.1% Tween 20 and 100 μl/well was added to the plate. Plates werethen incubated for 1 hour at room temperature. After washing, 1 mg/mlO-Phenylenediamine dihydrochloride solution (Sigma P9187) was preparedand 100 μl per well were added. The development was stopped with 50 μlper well 1M Hydrochloric acid (Fisher Chemical, J/4320/15) and theabsorbance at 490 nm was read on a Molecular Devices versamax platereader using Softmax (version 7.0). Titers were determined using theendpoint titer determination method. For each sample, an endpoint titeris defined as the reciprocal of the highest sample dilution that gives areading (OD) above the cut-off. The cut-off was determined for eachexperimental group as the mean OD+3SD of naïve samples.

Peripheral blood mononuclear cell isolation and resuscitation. PBMCswere isolated from whole blood anticoagulated with heparin (132 Unitsper 8 720 ml blood) (BD Biosciences, Oxford, UK) using standard methods.PBMCs isolated from tissues were stored at −180° C. For resuscitationPBMCs were thawed, washed in R10 medium (consisting of RPMI 1640supplemented with 2 mM L-glutamine, 50 U/ml penicillin-50 μg/mlstreptomycin, and 10% heat-inactivated FBS) with 1 U/ml of DNase(Sigma), and resuspended in R10 medium and incubated at 37° C. 5% CO₂overnight.

ELISpot. An IFNγ ELISpot assay was used to estimate the frequency andIFNγ production capacity of SARS-CoV-2-specific T cells in PBMCs using ahuman/simian IFNγ kit (MabTech, Nacka. Sweden), as described previously[Sibley, L. S., et al., ELISPOT Refinement Using Spot Morphology forAssessing Host Responses to Tuberculosis. Cells, 2012. 1(1): p. 5-141The cells were assayed at 2×10⁵ cells per well. Cells were stimulatedovernight with SARS-CoV-2 peptide pools spanning the ECD spike protein.Five peptide pools were 748 used, comprising of 15mer peptides,overlapping by 9 amino acids. Phorbol 12-myristate (Sigma) (100 ng/ml)and ionomycin (CN Biosciences, 753 Nottingham, UK) (1 mg/ml) were usedas a positive control. Results were calculated and reported as spotforming units (SFU) per million cells. All SARS-CoV-2 peptides wereassayed in duplicate and media only wells subtracted to give theantigen-specific SFU. ELISPOT plates were analyzed using a CTL scannerand software (CTL, Germany) and further analysis carried out usingGraphPad Prism (GraphPad Software, USA).

Statistics. All statistical analyses were performed using GraphPad Prism7 or 8 software (La Jolla, CA). These data were considered significantif p<0.05. The type of statistical analysis performed is detailed in thefigure legend. No samples or animals were excluded from the analysis.

Results:

Immunogenicity of one and two dose regimens of INO-4800. Twelve (6 maleand 6 female) rhesus macaques were vaccinated with 1 dose (6 animals) or2 doses (6 animals) of INO-4800 on day 28 or 0 and 28, respectively(FIG. 22A). For each treatment 1 mg INO-4800 was administeredintradermally followed by CELLECTRA-ID EP. A further six age- andsex-matched animals were not vaccinated and provided the control group.Animals were observed and scored as alert and healthy for the durationof the study, and no adverse events or clinical anomalies were recordedin the animals (FIG. 23 ). The serum titers of SARS-CoV-2 spike antigenreactive IgG antibodies in all animals were measured biweekly betweendays 0 and 56. In the single dose group (INO-4800 X1) a mean endpointtiter of 467 against the SARS-CoV-2 spike antigen trimeric 51+S2 ECDform and 442 against the RBD antigen, and a live virus (Victoria/01/2020matched to the challenge strain) neutralization titer of 239 14 daysafter vaccination (FIGS. 22B, 22C, 22D). In the 2 dose group (INO-4800X2) a mean endpoint titer of 2,142 against the 51+S2 ECD and 1,538against the RBD antigen, and a live virus neutralization titer of 2,199was measured 14 days after the 2nd vaccination (FIGS. 22B, 22C, 22D).Vaccination with INO-4800 induced SARS-CoV-2 spike antigen-specific Th1T cell responses in the PBMC population as measured by an IFN-γ ELISpot(FIG. 22E). In summary, intradermal delivery of INO-4800 induced afunctional humoral and T cell response against SARS-CoV-2 spike proteinwhich was boosted after a second dose. At the day of viral challenge(Day 56) the level of SARS-CoV-2 neutralizing antibodies in the serumwas significantly higher in the vaccinated groups compared to thecontrol group (p=0.015). Following viral challenge there was a slightincrease in SARS-CoV-2 spike binding and neutralizing antibody titers inall the groups between days 56 and 62-64 (FIGS. 22B, 22C, 22D). In thecontrol group there was an increase in the cellular immune response topeptides spanning the SARS-CoV-2 spike antigen after viral challenge,but little change in the vaccinated groups, likely due to control ofviral infection by the humoral arm of the host immune system (FIG. 22F).

Viral loads in the upper and lower respiratory tracts after SARS-CoV-2challenge

On day 56 all animals were challenged with a total of 5×10{circumflexover ( )}6 pfu SARS-CoV-2 delivered to both the upper and lowerrespiratory tract. No overt clinical symptoms were observed throughoutthe duration (6-8 days) of the challenge in any of the animals (FIGS.23A-23C). At indicated timepoints nasal and throat swabs were collectedfrom the animals. SARS-CoV-2 viral genomic (viral RNA) and subgenomic(sgmRNA), which represents replicating virus were measured by RT-qPCR(FIGS. 24A and 25A). Analysis of viral RNA area under the curve (AUC)levels in the throat revealed significantly reduced levels in thevaccinated groups (FIG. 24B). Additionally, the peak viral load levelmeasured in the INO-4800 X2 group was significantly reduced compared tothe control group (FIG. 24C). Analysis revealed a significant negativecorrelation between throat viral loads and neutralizing and anti-RBD IgGtiters (FIGS. 15A-15D). SARS-CoV-2 sgmRNA data revealed a similar trendto reduction of viral load in the vaccinated groups compared to controls(FIGS. 25A-25C). Analysis in the nasal compartment revealed a trend forreduction and accelerated clearance of viral RNA and sgmRNA in thevaccinated groups compared to control, but did not reach a level ofsignificance (FIGS. 24D-F and 25D-F). Analysis revealed a significantnegative correlation between nasal viral loads and neutralizing andanti-RBD IgG titers on day 3, but not day 1 (FIGS. 15E-15H).

At the time of necropsy (6-8 days post challenge), BAL fluid wascollected from each animal. Measurement of the levels of SARS-CoV-2viral RNA and sgmRNA revealed a reduction of the average virus invaccinated groups, even though the levels were variable within eachgroup dependent on the day of necropsy (FIGS. 26A, 26B). RT-qPCR wasalso performed on tissues collected at necropsy. At these timepointspost challenge the SARS-CoV-2 viral RNA levels detected were below limitof quantification in most tissues except the lungs (FIG. 27 ).Measurements of the level of SARS-CoV-2 viral mRNA and sgmRNA detectedin the lung tissue samples indicated reduced average viral load in thevaccinated animals (FIGS. 26C and 26D).

In summary data showed a significant reduction of viral load in thethroat, and a trend for a reduction of viral loads in the lungs of thevaccinated groups. The collection of BAL and lung tissue samples atdifferent timepoints (days 6, 7 or 8) after challenge likely added tothe intragroup variability observed impacting statistical analysis.RT-qPCR viral load data indicate INO-4800 vaccination has a positiveeffect in reducing viral loads in rhesus macaques challenged with highdose SARS-CoV-2, in general, lower viral levels were measured in the 2dose vaccine group compared to one dose vaccine group.

Disease Burden in the Lungs after SARS-CoV-2 Challenge.

The pulmonary disease burden was assessed on harvested lung tissuescollected at necropsy 6 to 8 days after challenge. Analysis wasperformed on all animals in the study in a double blinded manner.Histopathological analysis of lung tissue was performed on multipleorgan tissues, but only the lungs showed remarkable lesions, compatiblewith SARS-CoV-2 infection. Pulmonary lesions consistent with infectionwith SARS-CoV-2 were observed in the lungs of animals from theunvaccinated control and at a reduced level in vaccinated groups.Representative histopathology images are provided in FIG. 28 . Briefly,the lung parenchyma was comprised of multifocal to coalescing areas ofpneumonia surrounded by unaffected parenchyma. Alveolar damage, withnecrosis of pneumocytes was a prominent feature in the affected areas.Alveolar spaces and interalveolar septa contained mixed inflammatorycells (including macrophages, lymphocytes, viable and degeneratedneutrophils, and occasional eosinophils), and edema. Type II pneumocytehyperplasia was also observed in distal bronchioles andbronchiolo-alveolar junctions. In the larger airways occasional, focal,epithelial degeneration and sloughing was observed in the respiratoryepithelium. Low numbers of mixed inflammatory cells, comprisingneutrophils, lymphoid cells, and occasional eosinophils, infiltratedbronchial and bronchiolar walls. In the lumen of some airways, mucusadmixed with degenerative cells, mainly neutrophils and epithelialcells, was seen. Within the parenchyma, perivascular and peribronchiolarcuffing was also observed, being mostly lymphoid cells comprising theinfiltrates.

The histopathology score and percent tissue area of SARS-CoV-2 RNApositivity were applied to quantify the disease burden. The unvaccinatedgroup showed the highest histopathological scores in the lung whencompared with the vaccinated groups (FIGS. 29A and 29C). Animals fromvaccinated groups showed reduced pathology when compared withunvaccinated animals, except for animal #10A from INO-4800X1 group,which showed histopathological scores similar to the unvaccinatedanimals. To detect the presence of SARS-CoV-2 RNA in the lung tissue, insitu hybridization (ISH) was performed. Viral RNA was observed inpneumocytes and inflammatory cells within the histopathological lesionswith reduced frequency in the vaccinated animals (FIG. 29B).

CT scans were performed to provide an in-life, unbiased, andquantifiable metric of lung disease. Results from lung CT imagingperformed 5 days after challenge with SARS-CoV-2 were evaluated for thepresence of COVID-19 disease features: ground glass opacity (GGO),consolidation, crazy paving, nodules, pen-lobular consolidation;distribution—upper, middle, lower, central 2/3, peripheral,bronchocentric, and for pulmonary embolus. The medical radiologist wasblinded to the animal's treatment and clinical status. The extent oflung involvement was evaluated and quantified using a scoring systemdeveloped for COVID disease. The score system parameters are provided inmaterials and methods section. Pulmonary abnormalities characteristic ofCOVID-19 disease where observed in 3 out of 6 and 2 out of 6 animals inthe INO-4800 one dose or two dose groups, respectively, and in 5 out of6 unvaccinated animals in the control group (representative CT scanimages are provided in FIG. 30 ). The extent of lung involvement in theanimals with disease involvement was less than 25% and considered lowlevel disease (FIG. 29D). There was a trend for disease scores to behighest in the unvaccinated control group with a reduction in theINO-4800 one and two dose groups (FIGS. 29E-29G). The comparison ofscores between groups did not reach statistical difference (p=0.0584between INO-4800 two dose group and no vaccine group, Mann Whitneytest). One outlier animal (10A) in the INO-4800 X1 group scored higherthan other animals. However, the level of disease was still consideredlow and comparable disease burden had been observed in other NHPSARS-CoV-2 challenge studies performed under the same conditions. Insummary, CT scanning provides a useful measure of SARS-CoV-2-induceddisease in rhesus macaques. Day 5 post SARS-CoV-2 infection,abnormalities where present were reported at low levels (<25% of lunginvolved). Evidence from CT scans suggested trends for differences inpulmonary disease burden between groups, with disease burden highest inthe nonvaccinated control group.

In summary, after high dose SARS-CoV-2 challenge of nonhuman primatesthe disease burden was reduced in the animals receiving a single of twodose regimen of INO-4800 vaccine. There was no indication of vaccineenhanced disease, even in animals receiving a suboptimal one dosevaccination regimen.

Discussion

This example describes the safety, immunogenicity, and efficacyassessments of the SARS-CoV-2 DNA vaccine INO-4800 in a stringent highdose nonhuman primate challenge model. Intradermal delivery of 1 mg ofINO-4800 to rhesus macaques induces both humoral and T cell responsesagainst the SARS-CoV-2 spike antigen in both a 2-dose regimen and a 1dose regimen. Throughout the study no overt clinical events wererecorded in the animals. After a high dose SARS-CoV-2 challenge, areduction in viral loads was observed and lung disease burden in boththe 1 and 2 dose vaccine groups supporting the efficacy of INO-4800.Importantly, vaccine enhanced disease (VED) was not observed, even withthe 1 dose group.

The rhesus macaque model has become a widely employed model forassessing medical countermeasures against SARS-CoV-2. Importantly,wildtype non-adapted SARS-CoV-2 replicates in the respiratory tract ofrhesus macaques, and the animal presents with some of thecharacteristics observed in humans with mild COVID-19 symptoms[Salguero, F. J., et al., Comparison of Rhesus and Cynomolgus macaquesas an authentic model for COVID-19. 2020: p. 2020.09.17.301093;Mufioz-Fontela, C., et al., Animal models for COVID-19. Nature, 2020.586(7830): p. 509-515]. Here, focus was placed on the lung diseaseburden in SARS-CoV-2 challenged rhesus macaques which had beenvaccinated with INO-4800. While the level of lung disease burdenmeasured in the animals was mild, a significant reduction in ofhistopathology and viral detection scores in the lungs of vaccinatedanimals was observed (FIG. 29 ). This suggests the potential for apositive impact on the LRT disease which is observed in COVID-19patients which progress to severe disease. Interestingly, a significantreduction in viral loads in the throat compartment in the upperrespiratory tract was also observed, but only a trend for reduction inthe nasal compartment. It may be that differential induction of mucosalimmunity exists between the throat and nasal compartment. Interestingly,a significant negative correlation between the RBD targeting andneutralizing antibodies in the serum with throat, but not nasal, viralloads was observed at day 1 post challenge (FIG. 15 ). However, thelevels of these antibodies in either of these URT compartments were notassayed to provide further evidence of the presence of increased levelsof functional antibodies in the throat compared to nasal passage.Another possibility could be that viral control in the nasal compartmentwhere the extensive (5×10⁶ pfu) SARS-CoV-2 challenge dose was directlyinstilled may be a higher bar than in other mucosal compartments. Insupport of this, data in the control animals showed nasal swabs yieldedhigher viral titers than throat swabs, with similar observations beingreported in COVID-19 subjects [Mohammadi, A., et al., SARS-CoV-2detection in different respiratory sites: A systematic review andmeta-analysis. EBioMedicine, 2020. 59: p. 102903.]

Importantly, the data indicated that enhanced respiratory disease (ERD)was not associated with INO-4800 immunization in either the 1 dose or 2dose regimen. In the INO-4800 X1 dose group, one animal (10A) didpresent with the highest lung histopathology score and CT scan score.However, the multifocal lesions in animal 10A showed a similarhistopathological pattern as those observed in the animals from thenonvaccinated group, with no apparent influx of different inflammatorycell subpopulations in the infiltrates. A potential hallmark of vaccineenhanced disease is the increased influx of inflammatory cells such aseosinophils [Bolles, M., et al., A double-inactivated severe acuterespiratory syndrome coronavirus vaccine provides incomplete protectionin mice and induces increased eosinophilic proinflammatory pulmonaryresponse upon challenge. J Virol, 2011. 85(23): p. 12201-15; Yasui, F.,et al., Prior Immunization with Severe Acute Respiratory Syndrome(SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein CausesSevere Pneumonia in Mice Infected with SARS-CoV. The Journal ofImmunology, 2008. 181(9): p. 6337-6348.]. The CT scan and histopathologydata for animal 10A are believed not to be associated with ERD, butrather a disease score and pattern similar to that of nonvaccinatedanimals. Similar lung histopathology inflammation scores ranging fromminimal-mild to mild-moderate were reported in samples analyzed 7 or 8days after challenge in rhesus macaques receiving other vaccinecandidates [Corbett, K. S., et al., Evaluation of the mRNA-1273 Vaccineagainst SARS-CoV-2 in Nonhuman Primates. New England Journal ofMedicine, 2020. 383(16): p. 1544-1555]. Currently, VED remains atheoretical concern with SARS-CoV-2 vaccination and attempts to induceenhanced disease using a formalin inactivated whole virus preparation ofSARS-CoV-2 have failed to repeat the lung pathology previously reportedfor other inactivated respiratory viral vaccines [Bewley, K. R., et al.,Immunological and pathological outcomes of SARS-CoV-2 challenge afterformalin-inactivated vaccine immunization of ferrets and rhesusmacaques. 2020: p. 2020.12.21.423746].

This data compliments the NHP SARS-CoV-2 challenge study whichdemonstrated reduction in LRT viral loads several months after INO-4800immunization (Example 9). However, there are distinct differencesbetween the studies, including different doses and variants used for thechallenge stock, and the timing of the challenge. In the study describedin this example, the animal was challenged four weeks after the lastvaccination, at a timepoint when high levels of circulating neutralizingantibodies were present. In the other study, the level of serumSARS-CoV-2 neutralizing antibody was low at the time of challenge,protection appeared to be dependent on the recall of a memory response,with a strong humoral and cellular response against SARS-CoV-2 spikeantigen detected in the animals. Here, an anamnestic response of asimilar magnitude was not observed, suggesting protection may have beenmediated by the antibodies present in circulation at time of challengewhich is supported by the correlation between serum SARS-CoV-2 targetingantibody levels and reductions in viral loads (FIG. 15 ).

In conclusion, the results here in a stringent preclinical SARS-CoV-2animal model provide further support for the efficacy and safety of theDNA vaccine INO-4800 as a prophylactic countermeasure against COVID-19.Importantly, tested as a single dose immunization we observed a positiveimpact on the lung disease burden and no VED. Taken together withINO-4800 clinical data, INO-4800 has many attributes in terms of safety,efficacy and logistical feasibility due its high stability, negating theneed for challenging cold chain distribution requirements for globalaccess. Furthermore, synthetic DNA vaccine technology is amenable tohighly accelerated developmental timelines, permitting rapid design andtesting of candidates against new SARS-CoV-2 variants which displaypotential for immune escape [Wibmer, C. K., et al., SARS-CoV-2 501Y.V2escapes neutralization by South African COVID-19 donor plasma. 2021: p.2021.01.18.427166.; Moore, J. P. and P. A. Offit, SARS-CoV-2 Vaccinesand the Growing Threat of Viral Variants. JAMA, 20211

Example 9 SARS-COV-2 DNA Vaccine Induces Humoral and Cellular ImmunityResulting in Memory Responses which Provide Anamnestic Protection in aRhesus Macaque Challenge

The immunogenicity of a synthetic DNA vaccine encoding the SARS-CoV-2Spike protein was previously demonstrated in both mice and guinea pigs(Example 1). In this example, the durability of INO-4800-induced immuneresponses in rhesus macaques is demonstrated. ID-EP administration inrhesus macaques induced cellular and humoral responses to SARS-Cov-2 Sprotein, with additional cross reactivity to the SARS-CoV-1 S protein.Protective efficacy is demonstrated more than 3 months post-finalimmunization, demonstrating establishment of amamnestic immune responsesand reduced viral loads in vaccinated macaques. After viral challenge, areduction in subgenomic messenger RNA (sgmRNA) BAL viral loads wasobserved compared to control animals with 1 mg (1/5th the DNA dose)administered via intradermal (ID) delivery. This was associated withinduction of a rapid recall response in both cellular and humoral immunearms, supporting the potential for the INO-4800 candidate to moderatedisease. No adverse events or evidence of vaccine enhanced disease (VED)were observed in animals in the vaccine group. Reduced viral subgenomicRNA loads in the lower lung and lower VL were observed. In the nose, atrend of lower VL was observed. These data support that immunizationwith this DNA vaccine candidate limits active viral replication and hasthe potential to reduce severity of disease, as well as reduced viralshedding in the nasal cavity.

It is important to note that the initial viral loads detected in controlanimals in this study were on average 1-2 logs higher (10⁹ PFU/swab in4/5 NHPs on day 1 post-challenge) than in similar published studiesperformed under identical conditions (˜10⁷ PFU/swab) (Yu et al., 2020,Science, eabc6284). Only two of the prior reported NHP studies includedintranasal delivery as inoculation route for challenge (van Doremalen etal., 2020, bioRxiv 2020.05.13.093195; Yu et al., 2020, Science,eabc6284). High-dose challenge inoculums are frequently employed toensure take of infection, however non-lethal systems such as thisSARS-CoV-2 rhesus macaque model may artificially reduce the impact ofpotentially protective vaccines and interventions (Durudas et al., 2011,Curr HIV Res 9, 276-288; Innis et al., 2019, Vaccine 37, 4830-4834).Despite these limitations, this study demonstrated significant reductionin peak BAL sgmRNA and overall viral RNA, likely induced by rapidinduction of immunological memory mediated by both B and T cellcompartments. Wolfel et al reported nasal titers in patients average6.5×10⁵ copies/swab days 1-5 following onset of symptoms (Wolfel et al.,2020, Nature 581, 465-469). These titers are significantly lower thanthe challenge dose and support potential for the vaccine candidate tocontrol early during SARS-CoV-2 infection.

This study shows that DNA vaccination with a vaccine candidate targetingthe full-length SARS-CoV-2 spike protein likely increases theavailability T cell immunodominant epitopes leading to a broader andmore potent immune response, compared to partial domains and truncatedimmunogens. In this study, T cell cross-reactivity was observed toSARS-CoV-1.

In addition to T cells, INO-4800 induced durable antibody responses thatrapidly increased following SARS-CoV-2 challenge. It is furtherdemonstrated that INO-4800 induced robust neutralizing antibodyresponses against both D614 and G614 SARS-CoV-2 variants. While the D/G614 site is outside the RBD, it has been suggested that this shift hasthe potential to impact vaccine-elicited antibodies (Korber B et al.,2020, Cell 182:1-16). Other studies report that the G614 variantexhibits increased SARS-CoV-2 infectivity (Hu et al., 2020, bioRxiv2020.06.20.161323; Ozono S, 2020, bioRxiv 2020.06.15.151779). The datashows induction of comparable neutralization titers between D614 andG614 variants and that these responses are similarly recalled followingSARS-CoV-2 challenge.

Materials & Methods

Non-Human Primate Immunizations, IFNγ ELISpot and ELISA

DNA vaccine, INO-4800: The highly optimized DNA sequence encodingSARS-CoV-2 IgE-spike was created using Inovio's proprietary in silicoGene Optimization Algorithm to enhance expression and immunogenicity(Smith et al., 2020, Nat Commun 11, 2601). The optimized DNA sequencewas synthesized, digested with BamHI and XhoI, and cloned into theexpression vector pGX0001 under the control of the human cytomegalovirusimmediate-early promoter and a bovine growth hormone polyadenylationsignal.

Animals: All rhesus macaque experiments were approved by theInstitutional Animal Care and Use Committee at Bioqual (Rockville,Maryland), an Association for Assessment and Accreditation of LaboratoryAnimal Care (AAALAC) International accredited facility. Blood wascollected for blood chemistry, PBMC isolation, serological analysis. BALwas collected on Week 8 to assay lung antibody levels and on Days 1, 2,4, 7 post challenge to assay lung viral loads.

Immunizations, sample collection and viral challenge. Ten Chinese rhesusmacaques (ranging from 4.55 kg-5.55 kg) were randomly assigned in studyimmunized (3 males and 2 females) or naïve (2 males and 3 females).Immunized macaques received two 1 mg injections of SARS-CoV-2 DNAvaccine, INO-4800 at week 0 and 4 by ID-EP administration using theCELLECTRA 2000® Adaptive Constant Current Electroporation Device with a3P array (Inovio Pharmaceuticals). Blood was collected at indicated timepoints to analyse blood chemistry, peripheral blood mononuclear cells(PBMC) isolation, and serum was collected for serological analysis.Bronchoalveolar lavage was collected at Week 8 to assay lung antibodylevels. BAL from naïve animals was run as control. At week 17, allanimals were challenged with 1.2×10⁸ VP (1.1×10⁴ PFU) SARS-CoV-2. Viruswas administered as 1 ml by the intranasal (IN) route (0.5 ml in eachnostril) and 1 ml by the intratracheal (IT) route.

Peripheral blood mononuclear cell isolation. Blood was collected fromeach macaque into sodium citrate cell preparation tubes (CPT, BDBiosciences). The tubes were centrifuged to separate plasma andlymphocytes, according to the manufacturer's protocol. Samples weretransported by same-day shipment on cold-packs from Bioqual to TheWistar Institute for PBMC isolation. PBMCs were washed and residual redblood cells were removed using ammonium-chloride-potassium (ACK) lysisbuffer. Cells were counted using a ViCell counter (Beckman Coulter) andresuspended in RPMI 1640 (Corning), supplemented with 10% fetal bovineserum (Atlas), and 1% penicillin/streptomycin (Gibco). Fresh cells werethen plated for IFNγ ELISpot Assays and flow cytometry.

IFN-γ Enzyme-linked immunospot (ELISpot). Monkey interferon gamma(IFN-γ) ELISpot assay was performed to detect cellular responses. MonkeyIFN-γ ELISpotPro (alkaline phosphatase) plates (Mabtech, Sweeden, Cat#3421M-2APW-10) were blocked for a minimum of 2 hours with RPMI 1640(Corning), supplemented with 10% FBS and 1% penn/strep (R10). FollowingPBMC isolation, 200 000 cells from macaques were added to each well inthe presence of 1) overlapping peptide pools (15-mers with 9-meroverlaps) corresponding to the SARS-CoV-1, SARS-CoV-2, or MERS-CoV Spikeproteins (5 μg/mL/well final concentration), 2) R10 with DMSO (negativecontrol), 3) or anti-CD3 positive control (Mabtech, 1:1000 dilution).All samples were plated in triplicate. Plates were incubated overnightat 37° C., 5% CO₂. After 18-20 hours, the plates were washed in PBS andspots were developed according to the manufacturer's protocol. Spotswere imaged using a CTL Immunospot plate reader and antigen-specificresponses were determined by subtracting the number of spots in theR10+DMSO negative control well from the wells stimulated with peptidepools.

Antigen Binding ELISA. Serum and BAL was collected at each time pointwas evaluated for binding titers as indicated. Ninety-six wellimmunosorbent plates (NUNC) were coated with 1 ug/mL recombinantSARS-CoV-2 S1+S2 ECD protein (Sino Biological 40589-VO8B1), S1 protein(Sino Biological 40591-VO8H), S2 protein (Sino Biological 40590-VO8B),or receptor-binding domain (RBD) protein (Sino Biological 40595-VO5H) inDPBS overnight at 4° C. ELISA plates were also coated with 1 ug/mLrecombinant SARS-CoV S1 protein (Sino Biological 40150-V08B1) and RBDprotein (Sino Biological 40592-VO8B) or MERS-CoV Spike (Sino Biological40069-VO8B). Plates were washed four times with PBS+0.05% Tween20(PBS-T) and blocked with 5% skim milk in PBS-T (5% SM) for 90 minutes at37° C. Sera or BAL from INO-4800 vaccinated macaques were seriallydiluted in 5% SM, added to the washed ELISA plates, and incubated for 1hour at 37° C. Following incubation, plates were washed 4 times withPBS-T and an anti-monkey IgG conjugated to horseradish peroxidase(Southern Biotech 4700-5). Plates were washed 4 times with PBS-T andone-step TMB solution (Sigma) was added to the plates. The reaction wasstopped with an equal volume of 2N sulfuric acid. Plates were read at450 nm and 570 nm within 30 minutes of development using a BiotekSynergy2 plate reader.

ACE2 Competition ELISA-Non-human primates. 96-well half area plates(Corning) were coated at room temperature for 3 hours with 1 pg/mLPolyRab anti-His antibody (ThermoFisher, PA1-983B), followed byovernight blocking with blocking buffer containing 1×PBS, 5% skim milk,1% FBS, and 0.2% Tween-20. The plates were then incubated with 10 pg/mLof His6×-tagged SARS-CoV-2 (“His6×” disclosed as SEQ ID NO: 25), S1+S2ECD (Sinobiological, 40589-V08B1) at room temperature for 1-2 hours. NHPsera (Day 0 or Week 6) was serially diluted 3-fold with 1×PBS containing1% FBS and 0.2% Tween and pre-mixed with huACE2-IgMu at constantconcentration of 0.4 ug/ml. The pre-mixture was then added to the plateand incubated at RT for 1-2 hours. The plates were further incubated atroom temperature for 1 hour with goat anti-mouse IgG H+L HRP (A90-116P,Bethyl Laboratories) at 1:20,000 dilution followed by addition ofone-step TMB ultra substrate (ThermoFisher) and then quenched with 1MH₂SO₄. Absorbance at 450 nm and 570 nm were recorded with BioTEK platereader.

Flow cytometry-based ACE2 receptor binding inhibition assay. HEK-293Tcells stably expressing ACE2-GFP were generated using retroviraltransduction. Following transduction, the cells were flow sorted basedon GFP expression to isolate GFP positive cells. Single cell cloning wasdone on these cells to generate cell lines with equivalent expression ofACE2-GFP. To detect inhibition of Spike binding to ACE2, S1+S2 ECD-histagged (Sino Biological, Catalog #40589-VO8B1) was incubated with serumcollected from vaccinated animals at indicated time points and dilutionsat concentration of 2.5 μg/ml on ice for 60 minutes. This mixture wasthen transferred to 150,000 293T-ACE2-GFP cells and incubated on ice for90 minutes. Following this, the cells were washed 2× with PBS followedby staining for Surelight® APC conjugated anti-his antibody (Abcam,ab72579) for 30 min on ice. As a positive control, Spike protein waspre-incubated with recombinant human ACE2 before transferring to293T-Ace2-GFP cells. Data was acquired using a BD LSRII and analyzed byFlowJo (version 10).

Pseudovirus Neutralization Assay. SARS-CoV-2 pseudovirus were producedusing HEK293T cells transfected with GeneJammer (Agilent) usingIgE-SARS-CoV-2 S plasmid (Genscript) and pNL4-3.Luc.R-E- plasmid (NIHAIDS reagent) at a 1:1 ratio. Forty-eight hours post transfection,transfection supernatant was collected, enriched with FBS to 12% finalvolume, steri-filtered (Millipore Sigma), and aliquoted for storage at−80° C. SARS-Cov-2 pseudovirus neutralization assay was set up using D10media (DMEM supplemented with 10% FBS and 1× Penicillin-Streptomycin) ina 96 well format. CHO cells stably expressing Ace2 were used as targetcells (Creative Biolabs, Catalog No. VCeL-Wyb019). SARS-Cov-2pseudovirus were titered to yield greater than 20 times the cells onlycontrol relative luminescence units (RLU) after 72h of infection. Forsetting up neutralization assay, 10,000 CHO-ACE2 cells were plated in96-well plates in 100 ul D10 media and rested overnight at 37° C. and 5%CO2 for 24 hours. Following day, Monkey and Rabbit sera from INO-4800vaccinated and control groups were heat inactivated and serially dilutedas desired. Sera were incubated with a fixed amount of SARS-Cov-2pseudovirus for 90 minutes at RT. 50u1 media was removed from the platedCHO-Ace2 cell containing wells. Post 90 minutes, the mix was added toplated CHO-Ace2 cells and allowed to incubate in a standard incubator(37% humidity, 5% CO2) for 72h. Post 72h, cells were lysed usingbritelite plus luminescence reporter gene assay system (Perkin ElmerCatalog no. 6066769) and RLU were measured using the Biotek platereader. Neutralization titers (ID50) were calculated using GraphPadPrism 8 and defined as the reciprocal serum dilution at which RLU werereduced by 50% compared to RLU in virus control wells after subtractionof background RLU in cell control wells.

Viral RNA assay. RT-PCR assays were utilized to monitor viral loads,essentially as previously described (Abnink P et al 2019 Science).Briefly, RNA was extracted using a QIAcube HT (Qiagen, Germany) and theCador pathogen HT kit from bronchoalveolar lavage (BAL) supernatant andnasal swabs. RNA was reverse transcribed using superscript VILO(Invitrogen) and ran in duplicate using the QuantStudio 6 and 7 FlexReal-Time PCR System (Applied Biosystems) according to manufacturer'sspecifications. Viral loads were calculated of viral RNA copies per mLor per swab and the assay sensitivity was 50 copies. The target foramplification was the SARS-CoV2 N (nucleocapsid) gene. The primers andprobes for the targets were: 2019-nCoV_N1-F:5′-GACCCCAAAATCAGCGAAAT-3′(SEQ ID NO:18); 2019-nCoV N1-R: 5′-TCTGGTTACTGCCAGTTGAATCTG-3′ (SEQ IDNO:19); 2019-nCoV_N1-P: 5′-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3′ (SEQ IDNO:20).

Subgenomic mRNA assay. SARS-CoV-2 E gene subgenomic mRNA (sgmRNA) wasassessed by RT-PCR using an approach similar to previously described(Wolfel R et al. 2020, Nature, 581, 465-469). To generate a standardcurve, the SARS-CoV-2 E gene sgmRNA was cloned into a pcDNA3.1expression plasmid; this insert was transcribed using an AmpliCap-Max T7High Yield Message Maker Kit (Cellscript) to obtain RNA for standards.Prior to RT-PCR, samples collected from challenged animals or standardswere reverse-transcribed using Superscript III VILO (Invitrogen)according to the manufacturer's instructions. A Taqman custom geneexpression assay (ThermoFisher Scientific) was designed using thesequences targeting the E gene sgmRNA (18). Reactions were carried outon a QuantStudio 6 and 7 Flex Real-Time PCR System (Applied Biosystems)according to the manufacturer's specifications. Standard curves wereused to calculate sgmRNA in copies per ml or per swab; the quantitativeassay sensitivity was 50 copies per ml or per swab.

Results

Induction of memory humoral and cellular immune responses in INO-4800immunized non-human primates. Non-human primates (NHP) are a valuablemodel in the development of COVID-19 vaccines and therapeutics as theycan be infected with wild-type SARS-CoV-2, and present with similarpathology to humans (Chandrashekar et al., 2020, Science, eabc4776; Qinet al., 2005, J Pathol 206, 251-259; Yao et al., 2014, J Infect Dis 209,236-242; Yu et al., 2020, Science, eabc6284). Rhesus macaques (n=5)received two immunizations of INO-4800 (1 mg), at Week 0 and Week 4(FIG. 33A). Naïve control animals (n=5) did not receive vaccine. Humoraland cellular immune responses were monitored for 15 weeks (˜4 months)following prime immunization for memory responses. All animalsseroconverted following a single INO-4800 immunization, with serum IgGtiters detected against the full-length S1+S2 extracellular domain(ECD), S1, S2, and RBD regions of the SARS-CoV-2 S protein (FIG. 33B andFIG. 33C). Cross-reactive antibodies were also detected against SARS-CoVS1 protein and RBD, but not MERS-CoV (FIG. 34 ). SARS-CoV-2-reactive IgGagainst the ECD and RBD were detected in bronchoalveolar lavage (BAL)washes at Week 8 following immunization (FIG. 34 ).

In serum samples of the animals SARS-CoV-2 pseudovirus neutralizationactivity was detected for >4 months following immunization (FIG. 33D),demonstrating memory titers comparable to those observed in otherreported acute protection studies in macaques (Gao et al., 2020, Science369, 77-81; Tian et al., 2020, Emerg Microbes Infect, 9:382-385; vanDoremalen et al., 2020, bioRxiv 2020.05.13.093195; Yu et al., 2020,Science, eabc6284) and reported for convalescent humans (Ni et al.,2020, Immunity 52, 971-977 e973; Robbiani et al., 2020, Nature,s41586-020-2456-9). During the course of the COVID-19 pandemic, a D614GSARS-CoV-2 spike variant has emerged that has potentially greaterinfectivity, and now accounts for >80% of new isolates (Korber B et al.,2020, Cell 182:1-16). There is concern that vaccines developed prior tothis variant's appearance may not neutralize the D614G virus. Therefore,neutralization against this new variant was evaluated using a modifiedpseudovirus expressing the G614 Spike protein (FIG. 33E). Similarneutralization ID50 titers were observed against both D614 and G614spikes, supporting induction of functional antibody responses byINO-4800 against the now dominating SARS-CoV-2 variant.

To further investigate the neutralizing activities, the sera was alsotested using an ACE2 competition ELISA, where sera from 80% of immunizedNHPs inhibited the SARS-CoV-2 Spike-ACE2 interaction (FIG. 33F). 100% ofmacaques responded in the flow cytometry ACE2-293T inhibition assay,with 53-96% inhibition of the Spike-ACE2 interaction at a 1:10 dilutionand 24-53% inhibition at a 1:30 dilution (FIG. 33G).

INO-4800 immunization also induced SARS-CoV-2 S antigen reactive T cellresponses against all 5 peptide pools with T cells responses peaking atWeek 6, two weeks following the second immunization (0-518 SFU/millioncells) (FIG. 33H). Distinct immunogenic epitope responses were detectedagainst the RBD and S2 regions (FIG. 33B). Cross-reactive T cellsresponses were also detected against the SARS-CoV Spike protein (FIG.36A). However, cross-reactivity was not observed to MERS-CoV Spikepeptides, which supports the lower sequence homology between SARS-CoV-2and MERS-CoV (FIG. 36B).

Vaccine induced memory recall responses upon SARS-CoV-2 challenge innon-human primates. Vaccine immunized macaques along with unvaccinatedcontrols were challenged with SARS-CoV-2 13 weeks (˜3 months) post-finalimmunization (Study Week 17, FIG. 37A). NHPs received a challenge doseof 1.1×10⁴ PFU of SARS-CoV-2 Isolate USA-WA1/2020 by intranasal andintratracheal inoculation as previously described (Chandrashekar et al.,2020; Yu et al., 2020). Upon viral challenge, 3/5 of INO-4800 vaccinatedanimals had an immediate increase in antibody titers against theSARS-CoV-2 full-length ECD. By day 7, 5/5 animals had an increase inantibody titers against both full length ECD and RBD (FIG. 37B). Sevendays post-challenge, robust geometric mean endpoint titers ranging from409,600-1,638,400 were observed in immunized animals, compared with thenaïve group which displayed seroconversion of only 1/5 animals (GMT 100)(FIG. 37B). A significant increase in pseudoneutralization titers wasobserved in all INO-4800 immunized animals against both D614 and G614Spike variants by day 7 post-challenge (FIG. 37C).

Cellular responses were evaluated before and after challenge. At week15, IFN-γ ELISpot responses had contracted significantly since the peakresponses observed at week 6. T cell responses increased in thevaccinated group following challenge (218.36 SFU/million cells) implyingrecall of immunological T cell memory (FIG. 38 and FIG. 39 ).

Protective efficacy following SARS-CoV-2 challenge. At earlier timepoints post virus input at challenge, viral mRNA detection does notdiscriminate between input challenge inoculum and active infection,while sgmRNA levels are more likely representative of active cellularSARS-CoV-2 replication (Wolfel et al., 2020, Nature, 581, 465-469; Yu etal., 2020, Science, eabc6284). SARS-CoV-2 subgenomic mRNA (sgmRNA) wasmeasured in nonvaccinated control and INO-4800 vaccinated macaquesfollowing challenge with 1.1×10⁴ PFU of SARS-CoV-2 Isolate USA-WA1/2020(FIG. 40 ). Peak viral sgmRNA loads in the BAL were significantly lowerin the INO-4800 vaccinated group (FIG. 40A and FIG. 40B), along withsignificantly lower viral RNA loads at day 7 post-challenge (FIG. 40C),indicating protection from lower respiratory disease. While sgmRNA wasdetected in the nasal swabs of both the control and INO-4800 vaccinatedanimals (FIG. 40D through FIG. 40F), viral RNA levels trended downwardsin INO-4800 vaccinated animals by more than 2 logs (FIG. 40F). Overall,the reduced viral loads afforded by INO-4800 vaccination are likely dueto anamnestic B and T cell responses that are rapidly recalledimmediately following exposure to SARS-CoV-2 infection.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art. Such changes and modifications,including without limitation those relating to the chemical structures,substituents, derivatives, intermediates, syntheses, compositions,formulations, or methods of use of the invention, may be made withoutdeparting from the spirit and scope thereof.

ILLUSTRATIVE EMBODIMENTS

Embodiment 1. A nucleic acid molecule encoding a Severe AcuteRespiratory Syndrome coronavirus 2 (SARS-CoV-2) spike antigen, thenucleic acid molecule comprising:

-   -   a nucleic acid sequence having at least about 90% identity over        an entire length of the nucleic acid sequence set forth in        nucleotides 55 to 3837 of SEQ ID NO: 2;    -   a nucleic acid sequence having at least about 90% identity over        an entire length of SEQ ID NO: 2;    -   the nucleic acid sequence of nucleotides 55 to 3837 of SEQ ID        NO: 2;    -   the nucleic acid sequence of SEQ ID NO: 2;    -   a nucleic acid sequence having at least about 90% identity over        an entire length of SEQ ID NO: 3;    -   the nucleic acid sequence of SEQ ID NO: 3;    -   a nucleic acid sequence having at least about 90% identity over        an entire length of nucleotides 55 to 3837 of SEQ ID NO: 5;    -   a nucleic acid sequence having at least about 90% identity over        an entire length of SEQ ID NO: 5;    -   the nucleic acid sequence of nucleotides 55 to 3837 of SEQ ID        NO: 5;    -   the nucleic acid sequence of SEQ ID NO: 5;    -   a nucleic acid sequence having at least about 90% identity over        an entire length of SEQ ID NO: 6; or    -   the nucleic acid sequence of SEQ ID NO: 6.

Embodiment 2. A nucleic acid molecule encoding a SARS-CoV-2 spikeantigen, wherein the SARS-CoV-2 spike antigen comprises:

-   -   an amino acid sequence having at least about 90% identity over        an entire length of residues 19 to 1279 of SEQ ID NO: 1;    -   the amino acid sequence set forth in residues 19 to 1279 of SEQ        ID NO: 1;    -   an amino acid sequence having at least about 90% identity over        an entire length of SEQ ID NO: 1;    -   the amino acid sequence of SEQ ID NO: 1;    -   an amino acid sequence having at least about 90% identity over        an entire length of residues 19 to 1279 of SEQ ID NO: 4;    -   an amino acid sequence having at least about 90% identity over        an entire length of SEQ ID NO: 4;    -   the amino acid sequence set forth in residues 19 to 1279 of SEQ        ID NO: 4; or    -   the amino acid sequence of SEQ ID NO: 4.

Embodiment 3. An expression vector comprising the nucleic acid moleculeaccording to Embodiment 1 or Embodiment 2.

Embodiment 4. The expression vector according to Embodiment 3, whereinthe nucleic acid molecule is operably linked to a regulatory elementselected from a promoter and a poly-adenylation signal.

Embodiment 5. The expression vector according to Embodiment 3 orEmbodiment 4, wherein the vector is a plasmid or viral vector.

Embodiment 6. An immunogenic composition comprising an effective amountof the expression vector according to any one of Embodiments 3-5.

Embodiment 7. The immunogenic composition according to Embodiment 6further comprising a pharmaceutically acceptable excipient.

Embodiment 8. The immunogenic composition according to Embodiment 7wherein the pharmaceutically acceptable excipient comprises a buffer,optionally saline-sodium citrate buffer.

Embodiment 9. The immunogenic composition of Embodiment 8, wherein thecomposition is formulated at a concentration of 10 mg per milliliter ofa sodium salt citrate buffer.

Embodiment 10. The immunogenic composition according to any one ofEmbodiments 6-9, further comprising an adjuvant.

Embodiment 11. A SARS-CoV-2 spike antigen comprising:

-   -   an amino acid sequence having at least about 90% identity over        an entire length of residues 19 to 1279 of SEQ ID NO: 1;    -   the amino acid sequence set forth in residues 19 to 1279 of SEQ        ID NO: 1;    -   an amino acid sequence having at least about 90% identity over        an entire length of SEQ ID NO: 1;    -   the amino acid sequence of SEQ ID NO: 1;    -   an amino acid sequence having at least about 90% identity over        an entire length of residues 19 to 1279 of SEQ ID NO: 4;    -   an amino acid sequence having at least about 90% identity over        an entire length of SEQ ID NO: 4;    -   the amino acid sequence set forth in residues 19 to 1279 of SEQ        ID NO: 4; or    -   the amino acid sequence of SEQ ID NO: 4.

Embodiment 12. A vaccine for the prevention or treatment of Severe AcuteRespiratory Syndrome coronavirus 2 (SARS-CoV-2) infection comprising aneffective amount of the nucleic acid molecule of Embodiment 1 or 2, thevector of any one of Embodiments 3-5, or the antigen of Embodiment 11.

Embodiment 13. The vaccine according to Embodiment 12, furthercomprising a pharmaceutically acceptable excipient.

Embodiment 14. The vaccine according to Embodiment 13, wherein thepharmaceutically acceptable excipient comprises a buffer, optionallysodium salt citrate buffer.

Embodiment 15. The vaccine according to Embodiment 14, formulated at aconcentration of 10 mg of nucleic acid per milliliter of a sodium saltcitrate buffer.

Embodiment 16. The vaccine according to any one of Embodiments 12 to 15,further comprising an adjuvant.

Embodiment 17. A method of inducing an immune response against SevereAcute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) in a subject inneed thereof, the method comprising administering an effective amount ofthe nucleic acid molecule of Embodiment 1 or 2, the vector of any one ofEmbodiments 3-5, the immunogenic composition of any one of

Embodiments 6-10, the antigen of Embodiment 11, or the vaccine of anyone of Embodiments 12-16 to the subject.

Embodiment 18. A method of protecting a subject in need thereof frominfection with Severe Acute Respiratory Syndrome coronavirus 2(SARS-CoV-2), the method comprising administering an effective amount ofthe nucleic acid molecule of Embodiment 1 or 2, the vector of any one ofEmbodiments 3-5, the immunogenic composition of any one of Embodiments6-10, the antigen of Embodiment 11, or the vaccine of any one ofEmbodiments 12-16 to the subject.

Embodiment 19. A method of protecting a subject in need thereof from adisease or disorder associated with infection with Severe AcuteRespiratory Syndrome coronavirus 2 (SARS-CoV-2), the method comprisingadministering an effective amount of the nucleic acid molecule ofEmbodiment 1 or 2, the vector of any one of Embodiments 3-5, theimmunogenic composition of any one of Embodiments 6-10, the antigen ofEmbodiment 11, or the vaccine of any one of Embodiments 12-16 to thesubject.

Embodiment 20. A method of treating a subject in need thereof againstSevere Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infection,the method comprising administering an effective amount of the nucleicacid molecule of Embodiment 1 or 2, the vector of any one of Embodiments3-5, the immunogenic composition of any one of Embodiments 6-10, theantigen of Embodiment 11, or the vaccine of any one of Embodiments 12-16to the subject, wherein the subject is thereby resistant to one or moreSARS-CoV-2 strains.

Embodiment 21. The method of any one of Embodiments 17 to 20, whereinadministering comprises at least one of electroporation and injection.

Embodiment 22. The method of any one of Embodiments 17 to 20, whereinadministering comprises parenteral administration followed byelectroporation.

Embodiment 23. The method of any one of Embodiments 17 to 22, wherein aninitial dose of about 0.5 mg to about 2.0 mg of nucleic acid isadministered to the subject, optionally wherein the initial dose is 0.5mg, 1.0 mg or 2.0 mg of nucleic acid.

Embodiment 24. The method of Embodiment 23, wherein a subsequent dose ofabout 0.5 mg to about 2.0 mg of nucleic acid is administered to thesubject about four weeks after the initial dose, optionally wherein thesubsequent dose is 0.5 mg, 1.0 mg or 2.0 mg of nucleic acid.

Embodiment 25. The method of Embodiment 24, wherein one or more furthersubsequent doses of about 0.5 mg to about 2.0 mg of nucleic acid isadministered to the subject at least twelve weeks after the initialdose, optionally wherein the further subsequent dose is 0.5 mg, 1.0 mg,or 2.0 mg of nucleic acid.

Embodiment 26. The method of any one of Embodiments 17 to 25, comprisingadministering INO-4800 or a biosimilar thereof to the subject.

Embodiment 27. The method of any one of Embodiments 17 to 26, furthercomprising administering to the subject at least one additional agentfor the treatment of SARS-CoV-2 infection or the treatment or preventionof a disease or disorder associated with SARS-CoV-2 infection.

Embodiment 28. The method of Embodiment 27 wherein the nucleic acidmolecule, vector, the immunogenic composition, antigen, or vaccine isadministered to the subject before, concurrently with, or after theadditional agent.

Embodiment 29. Use of the nucleic acid molecule of Embodiment 1 or 2,the vector of any one of Embodiments 3-5, the immunogenic composition ofany one of Embodiments 6-10, the antigen of Embodiment 11, or thevaccine of any one of Embodiments 12-16 in a method of inducing animmune response against Severe Acute Respiratory Syndrome coronavirus 2(SARS-CoV-2) in a subject in need thereof.

Embodiment 30. Use of the nucleic acid molecule of Embodiment 1 or 2,the vector of any one of Embodiments 3-5, the immunogenic composition ofany one of Embodiments 6-10, the antigen of Embodiment 11, or thevaccine of any one of Embodiments 12-16 in a method of protecting asubject from infection with Severe Acute Respiratory Syndromecoronavirus 2 (SARS-CoV-2).

Embodiment 31. Use of the nucleic acid molecule of Embodiment 1 or 2,the vector of any one of Embodiments 3-5, the immunogenic composition ofany one of Embodiments 6-10, the antigen of Embodiment 11, or thevaccine of any one of Embodiments 12-16 in a method of protecting asubject from a disease or disorder associated with infection with SevereAcute Respiratory Syndrome coronavirus 2 (SARS-CoV-2).

Embodiment 32. Use of the nucleic acid molecule of Embodiment 1 or 2,the vector of any one of Embodiments 3-5, the immunogenic composition ofany one of Embodiments 6-10, the antigen of Embodiment 11, or thevaccine of any one of Embodiments 12-16 in a method of treating asubject in need thereof against Severe Acute Respiratory Syndromecoronavirus 2 (SARS-CoV-2) infection.

Embodiment 33. The use of any one of Embodiments 29 to 32 in combinationwith at least one additional agent for the treatment of SARS-CoV-2infection or the treatment or prevention of a disease or disorderassociated with SARS-CoV-2 infection.

Embodiment 34. The use of any one of Embodiments 29 to 33, wherein thenucleic acid molecule, the vector, the immunogenic composition, theantigen, or the vaccine is administered to the subject by at least oneof electroporation and injection.

Embodiment 35. The use of Embodiment 34, wherein the nucleic acidmolecule, the vector, the immunogenic composition, the antigen, or thevaccine is administered to the subject by parenteral administrationfollowed by electroporation.

Embodiment 36. The use of any one of Embodiments 29 to 35, wherein aninitial dose of about 0.5 mg to about 2.0 mg of nucleic acid isadministered to the subject, optionally wherein the initial dose is 0.5mg, 1.0 mg, or 2.0 mg of nucleic acid.

Embodiment 37. The use of Embodiment 36, wherein a subsequent dose ofabout 0.5 mg to about 2.0 mg of nucleic acid is administered to thesubject about four weeks after the initial dose, optionally wherein thesubsequent dose is 0.5 mg, 1.0 mg, or 2.0 mg of nucleic acid.

Embodiment 38. The use of Embodiment 37, wherein a further subsequentdose of about 0.5 mg to about 2.0 mg of nucleic acid is administered tothe subject at least twelve weeks after the initial dose, optionallywherein the further subsequent dose is 0.5 mg, 1.0 mg, or 2.0 mg ofnucleic acid.

Embodiment 39. The use of any one of Embodiments 29 to 38, wherein theimmunogenic composition is INO-4800 or a biosimilar thereof.

Embodiment 40. Use of the nucleic acid molecule of Embodiment 1 or 2,the vector of any one of Embodiments 3-5, or the antigen of Embodiment11 in the preparation of a medicament.

Embodiment 41. Use of the nucleic acid molecule of Embodiment 1 or 2,the vector of any one of Embodiments 3-5, or the antigen of Embodiment11 in the preparation of a medicament for treating or protecting againstinfection with Severe Acute Respiratory Syndrome coronavirus 2(SARS-CoV-2).

Embodiment 42. Use of the nucleic acid molecule of Embodiment 1 or 2,the vector of any one of Embodiments 3-5, or the antigen of Embodiment11 in the preparation of a medicament for protecting a subject in needthereof from a disease or disorder associated with infection with SevereAcute Respiratory Syndrome coronavirus 2 (SARS-CoV-2).

Embodiment 43. A method of detecting a persistent cellular immuneresponse in a subject, the method comprising the steps of:

-   -   administering an immunogenic composition for inducing an immune        response against a SARS-CoV-2 antigen to a subject in need        thereof;    -   isolating peripheral mononuclear cells (PBMCs) from the subject;    -   stimulating the isolated PBMCs with a SARS-CoV-2 spike antigen        comprising an amino acid sequence selected from the group        consisting of an amino acid sequence having at least about 90%        identity over an entire length of residues 19 to 1279 of SEQ ID        NO: 1, the amino acid sequence set forth in residues 19 to 1279        of SEQ ID NO: 1, an amino acid sequence having at least about        90% identity over an entire length of SEQ ID NO: 1, the amino        acid sequence of SEQ ID NO: 1, an amino acid sequence having at        least about 90% identity over an entire length of residues 19 to        1279 of SEQ ID NO: 4, an amino acid sequence having at least        about 90% identity over an entire length of SEQ ID NO: 4, the        amino acid sequence set forth in residues 19 to 1279 of SEQ ID        NO: 4, the amino acid sequence of SEQ ID NO: 4, and a fragment        thereof comprising at least 20 amino acids; and    -   detecting at least one of the number of cytokine expressing        cells and the level of cytokine expression.

Embodiment 44. The method of Embodiment 43, wherein the step ofdetecting at least one of the number of cytokine expressing cells andthe level of cytokine expression is performed using an assay selectedfrom the group consisting of Enzyme-linked immunospot (ELISpot) andIntracellular Cytokine Staining (ICS) analysis using flow cytometry.

Embodiment 45. The method of Embodiment 43, wherein the subject isadministered an immunogenic composition comprising a nucleic acidmolecule, wherein the nucleic acid molecule comprises a nucleotidesequence selected from the group consisting of:

-   -   a nucleic acid sequence having at least about 90% identity over        an entire length of the nucleic acid sequence set forth in        nucleotides 55 to 3837 of SEQ ID NO: 2;    -   a nucleic acid sequence having at least about 90% identity over        an entire length of SEQ ID NO: 2;    -   the nucleic acid sequence of nucleotides 55 to 3837 of SEQ ID        NO: 2;    -   the nucleic acid sequence of SEQ ID NO: 2;    -   a nucleic acid sequence having at least about 90% identity over        an entire length of SEQ ID NO: 3;    -   the nucleic acid sequence of SEQ ID NO: 3;    -   a nucleic acid sequence having at least about 90% identity over        an entire length of nucleotides 55 to 3837 of SEQ ID NO: 5;    -   a nucleic acid sequence having at least about 90% identity over        an entire length of SEQ ID NO: 5;    -   the nucleic acid sequence of nucleotides 55 to 3837 of SEQ ID        NO: 5;    -   the nucleic acid sequence of SEQ ID NO: 5;    -   a nucleic acid sequence having at least about 90% identity over        an entire length of SEQ ID NO: 6; and    -   the nucleic acid sequence of SEQ ID NO: 6.

SEQUENCE LISTING SARS-CoV-2 Consensus Spike Antigen amino acid insertsequence of pGX9501 (SEQ ID NO: 1) (IgE leader sequence underlined):   1 MDWTWILFLV AAATRVHSSQ CVNLTTRTQL PPAYTNSFTR GVYYPDKVFR SSVLHSTQDL  61 FLPFFSNVTW FHAIHVSGTN GTKRFDNPVL PFNDGVYFAS TEKSNIIRGW IFGTTLDSKT 121 QSLLIVNNAT NVVIKVCEFQ FCNDPFLGVY YHKNNKSWME SEFRVYSSAN NCTFEYVSQP 181 FLMDLEGKQG NFKNLREFVF KNIDGYFKIY SKHTPINLVR DLPQGFSALE PLVDLPIGIN 241 ITRFQTLLAL HRSYLTPGDS SSGWTAGAAA YYVGYLQPRT FLLKYNENGT ITDAVDCALD 301 PLSETKCTLK SFTVEKGIYQ TSNFRVQPTE SIVRFPNITN LCPFGEVFNA TRFASVYAWN 361 RKRISNCVAD YSVLYNSASF STFKCYGVSP TKLNDLCFTN VYADSFVIRG DEVRQIAPGQ 421 TGKIADYNYK LPDDFTGCVI AWNSNNLDSK VGGNYNYLYR LFRKSNLKPF ERDISTEIYQ 481 AGSTPCNGVE GENCYFPLQS YGFQPTNGVG YQPYRVVVLS FELLHAPATV CGPKKSTNLV 541 KNKCVNFNFN GLTGTGVLTE SNKKFLPFQQ FGRDIADTTD AVRDPQTLEI LDITPCSFGG 601 VSVITPGTNT SNQVAVLYQD VNCTEVPVAI HADQLTPTWR VYSTGSNVFQ TRAGCLIGAE 661 HVNNSYECDI PIGAGICASY QTQTNSPRRA RSVASQSIIA YTMSLGAENS VAYSNNSIAI 721 PTNFTISVTT EILPVSMTKT SVDCTMYICG DSTECSNLLL QYGSFCTQLN RALTGIAVEQ 781 DKNTQEVFAQ VKQIYKTPPI KDFGGFNFSQ ILPDPSKPSK RSFIEDLLFN KVTLADAGFI 841 KQYGDCLGDI AARDLICAQK FNGLTVLPPL LTDEMIAQYT SALLAGTITS GWTFGAGAAL 901 QIPFAMQMAY RENGIGVTQN VLYENQKLIA NQFNSAIGKI QDSLSSTASA LGKLQDVVNQ 961 NAQALNTLVK QLSSNFGAIS SVLNDILSRL DKVEAEVQID RLITGRLQSL QTYVTQQLIR1021 AAEIRASANL AATKMSECVL GQSKRVDFCG KGYHLMSFPQ SAPHGVVFLH VTYVPAQEKN1081 FTTAPAICHD GKAHFPREGV FVSNGTHWFV TQRNFYEPQI ITTDNTFVSG NCDVVIGIVN1141 NTVYDPLQPE LDSFKEELDK YFKNHTSPDV DLGDISGINA SVVNIQKEID RLNEVAKNLN1201 ESLIDLQELG KYEQYIKWPW YIWLGFIAGL IAIVMVTIML CCMTSCCSCL KGCCSCGSCC1261 KFDEDDSEPV LKGVKLHYT DNA insert sequence of pGX9501 (SEQ ID NO: 2)(IgE leader sequence underlined):   1 ATGGATTGGA CTTGGATTCT CTTTCTCGTT GCTGCAGCCA CACGCGTTCA TAGCAGCCAG  61 TGTGTGAACC TGACCACCAG AACACAGCTG CCTCCTGCCT ACACCAACAG CTTCACCAGA 121 GGAGTCTACT ACCCAGACAA AGTCTTCAGA AGCTCTGTGC TGCACAGCAC CCAGGACCTG 181 TTCCTGCCTT TCTTCAGCAA CGTGACCTGG TTCCACGCCA TCCACGTGTC TGGCACCAAC 241 GGCACCAAGA GATTTGACAA CCCTGTTCTT CCTTTCAATG ATGGCGTGTA CTTTGCCAGC 301 ACAGAGAAGA GCAACATCAT CCGAGGCTGG ATCTTTGGCA CCACCCTGGA CAGCAAAACC 361 CAGAGCCTGC TGATCGTGAA CAACGCCACC AACGTGGTCA TCAAGGTGTG TGAGTTCCAG 421 TTCTGCAATG ACCCTTTCCT GGGCGTGTAC TACCACAAGA ACAACAAGTC CTGGATGGAG 481 TCTGAGTTCA GAGTCTACAG CTCTGCCAAC AACTGCACAT TTGAATATGT GTCCCAGCCT 541 TTCCTGATGG ACCTGGAGGG CAAGCAGGGC AACTTTAAGA ACCTGAGAGA ATTTGTGTTC 601 AAGAACATCG ATGGCTACTT CAAGATCTAC AGCAAGCACA CACCCATCAA CCTGGTGAGA 661 GACCTGCCTC AGGGCTTCTC TGCCCTGGAG CCTCTGGTGG ACCTGCCCAT CGGCATCAAC 721 ATCACCAGAT TCCAGACCCT GCTGGCCCTG CACAGAAGCT ACCTGACCCC AGGAGACAGC 781 AGCAGCGGCT GGACAGCTGG AGCTGCTGCC TACTACGTGG GCTACCTGCA GCCCAGGACC 841 TTCCTGCTGA AGTACAACGA AAATGGCACC ATCACAGATG CTGTTGACTG TGCCCTGGAC 901 CCTCTTAGCG AGACCAAGTG CACCCTGAAG TCCTTCACAG TGGAGAAAGG CATCTACCAG 961 ACCAGCAACT TCCGAGTGCA GCCAACAGAG AGCATCGTGA GATTTCCAAA CATCACCAAC1021 CTGTGCCCTT TTGGAGAAGT CTTCAATGCC ACCAGATTTG CTTCTGTGTA CGCCTGGAAC1081 AGAAAAAGAA TCAGCAACTG TGTGGCTGAC TACTCTGTGC TGTACAACTC TGCCTCCTTC1141 TCCACCTTCA AGTGCTATGG AGTCTCTCCA ACCAAGCTGA ATGACCTGTG CTTCACCAAC1201 GTGTATGCTG ACAGCTTTGT GATCAGAGGA GATGAAGTGC GGCAGATTGC TCCTGGCCAG1261 ACAGGCAAGA TTGCTGACTA CAACTACAAG CTGCCTGATG ACTTCACAGG CTGTGTCATC1321 GCCTGGAACA GCAACAACCT GGACAGCAAG GTGGGCGGCA ACTACAACTA CCTGTACAGA1381 CTTTTCAGGA AGAGCAACCT GAAGCCTTTT GAAAGAGACA TCTCCACAGA GATCTACCAG1441 GCTGGCAGCA CACCCTGCAA TGGTGTGGAA GGCTTCAACT GCTACTTCCC TCTGCAGAGC1501 TACGGCTTCC AGCCAACAAA TGGCGTGGGC TACCAGCCTT ACAGAGTGGT GGTGCTGTCC1561 TTTGAGCTGC TGCACGCCCC TGCCACAGTG TGTGGCCCCA AGAAGAGCAC CAACCTGGTG1621 AAGAACAAAT GTGTGAACTT CAATTTCAAT GGCCTGACAG GCACAGGAGT GCTGACAGAG1681 AGCAACAAGA AGTTTCTTCC TTTCCAGCAG TTTGGAAGAG ACATTGCTGA CACCACAGAT1741 GCTGTGAGAG ATCCTCAGAC CCTGGAGATC CTGGATATCA CACCCTGCTC CTTTGGAGGA1801 GTTTCTGTCA TCACACCTGG CACCAATACC AGCAACCAAG TGGCTGTGCT GTACCAAGAT1861 GTGAATTGCA CAGAAGTGCC TGTGGCCATC CACGCTGACC AGCTGACACC CACCTGGAGA1921 GTGTACAGCA CAGGCAGCAA TGTTTTCCAG ACAAGAGCTG GCTGCCTGAT TGGAGCAGAG1981 CACGTGAACA ACAGCTATGA ATGTGACATC CCTATTGGAG CTGGCATCTG TGCCAGCTAC2041 CAGACCCAAA CCAACAGCCC AAGAAGAGCC AGATCTGTGG CCAGCCAGAG CATCATCGCC2101 TACACCATGA GCCTGGGAGC TGAGAACTCT GTGGCCTACA GCAACAACAG CATCGCCATC2161 CCCACCAACT TCACCATCTC TGTGACCACA GAGATCCTGC CTGTGTCCAT GACCAAGACA2221 TCTGTGGACT GCACCATGTA CATCTGTGGA GACAGCACAG AATGCAGCAA CCTGCTGCTG2281 CAGTACGGCT CCTTCTGCAC CCAGCTGAAC AGAGCCCTGA CAGGCATCGC TGTGGAGCAG2341 GACAAGAACA CACAGGAAGT GTTTGCCCAG GTGAAGCAGA TCTACAAAAC ACCACCCATC2401 AAGGACTTTG GAGGCTTCAA TTTCTCCCAA ATCCTGCCTG ACCCCAGCAA GCCTTCCAAG2461 AGAAGCTTCA TTGAAGACCT GCTGTTCAAC AAAGTGACCC TGGCTGATGC TGGCTTCATC2521 AAGCAGTATG GAGACTGCCT GGGAGACATT GCTGCCAGAG ACCTGATCTG TGCCCAGAAG2581 TTTAATGGCC TGACTGTGCT GCCTCCTCTG CTGACAGATG AAATGATCGC CCAGTACACA2641 TCTGCCCTGC TGGCTGGCAC CATCACCAGT GGCTGGACAT TTGGAGCTGG AGCTGCCCTG2701 CAGATCCCTT TTGCCATGCA GATGGCCTAC AGATTTAATG GCATCGGCGT GACCCAGAAC2761 GTGCTGTACG AGAACCAGAA GCTGATCGCC AACCAGTTCA ACTCTGCCAT CGGCAAGATC2821 CAGGACAGCC TGAGCAGCAC AGCCTCTGCC CTGGGCAAGC TGCAGGATGT GGTGAACCAA2881 AACGCCCAGG CCCTGAACAC CCTGGTGAAG CAGCTGAGCA GCAACTTTGG AGCCATCTCC2941 TCTGTGCTGA ATGACATCCT GAGCCGGCTG GACAAGGTGG AAGCAGAAGT GCAGATCGAC3001 AGACTCATCA CAGGCCGCCT GCAGAGCCTG CAGACCTACG TGACCCAGCA GCTGATCAGA3061 GCTGCTGAGA TCCGGGCCTC TGCCAACCTG GCTGCCACCA AGATGTCAGA ATGTGTGCTG3121 GGCCAGAGCA AAAGAGTGGA CTTCTGTGGC AAAGGCTACC ACCTGATGTC CTTCCCTCAG3181 TCTGCTCCTC ACGGCGTGGT GTTCCTGCAC GTGACCTACG TGCCTGCCCA GGAGAAGAAC3241 TTCACCACAG CTCCTGCCAT CTGCCACGAT GGCAAGGCCC ACTTCCCAAG AGAAGGTGTC3301 TTTGTGTCCA ATGGCACCCA CTGGTTCGTG ACCCAGAGAA ACTTCTACGA GCCTCAGATC3361 ATCACCACAG ACAACACATT TGTGTCTGGC AACTGTGATG TGGTCATCGG CATCGTGAAC3421 AACACAGTTT ATGACCCTCT GCAGCCTGAG CTGGACAGCT TCAAAGAAGA GCTGGACAAG3481 TACTTCAAGA ACCACACATC TCCAGATGTG GACCTGGGAG ACATCTCTGG CATCAATGCC3541 TCTGTGGTGA ACATCCAGAA GGAAATTGAC AGGCTGAACG AAGTGGCCAA GAACCTGAAC3601 GAAAGCCTCA TCGACCTGCA GGAGCTGGGC AAGTACGAGC AGTACATCAA GTGGCCTTGG3661 TACATCTGGC TGGGCTTCAT CGCTGGCCTC ATCGCCATCG TGATGGTGAC CATCATGCTG3721 TGCTGCATGA CCAGCTGCTG CTCTTGCCTG AAGGGCTGCT GCAGCTGTGG CAGCTGCTGC3781 AAGTTTGATG AAGATGACTC TGAGCCTGTG CTGAAGGGCG TGAAGCTGCA CTACACASingle strand DNA sequence of pGX9501 (SEQ ID NO: 3):   1 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta  61 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 121 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 181 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 241 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 301 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 361 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 421 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 481 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 541 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 601 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 661 aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 721 accgagctcg gatccgccac catggattgg acttggattc tctttctcgt tgctgcagcc 781 acacgcgttc atagcagcca gtgtgtgaac ctgaccacca gaacacagct gcctcctgcc 841 tacaccaaca gcttcaccag aggagtctac tacccagaca aagtcttcag aagctctgtg 901 ctgcacagca cccaggacct gttcctgcct ttcttcagca acgtgacctg gttccacgcc 961 atccacgtgt ctggcaccaa cggcaccaag agatttgaca accctgttct tcctttcaat1021 gatggcgtgt actttgccag cacagagaag agcaacatca tccgaggctg gatctttggc1081 accaccctgg acagcaaaac ccagagcctg ctgatcgtga acaacgccac caacgtggtc1141 atcaaggtgt gtgagttcca gttctgcaat gaccctttcc tgggcgtgta ctaccacaag1201 aacaacaagt cctggatgga gtctgagttc agagtctaca gctctgccaa caactgcaca1261 tttgaatatg tgtcccagcc tttcctgatg gacctggagg gcaagcaggg caactttaag1321 aacctgagag aatttgtgtt caagaacatc gatggctact tcaagatcta cagcaagcac1381 acacccatca acctggtgag agacctgcct cagggcttct ctgccctgga gcctctggtg1441 gacctgccca tcggcatcaa catcaccaga ttccagaccc tgctggccct gcacagaagc1501 tacctgaccc caggagacag cagcagcggc tggacagctg gagctgctgc ctactacgtg1561 ggctacctgc agcccaggac cttcctgctg aagtacaacg aaaatggcac catcacagat1621 gctgttgact gtgccctgga ccctcttagc gagaccaagt gcaccctgaa gtccttcaca1681 gtggagaaag gcatctacca gaccagcaac ttccgagtgc agccaacaga gagcatcgtg1741 agatttccaa acatcaccaa cctgtgccct tttggagaag tcttcaatgc caccagattt1801 gcttctgtgt acgcctggaa cagaaaaaga atcagcaact gtgtggctga ctactctgtg1861 ctgtacaact ctgcctcctt ctccaccttc aagtgctatg gagtctctcc aaccaagctg1921 aatgacctgt gcttcaccaa cgtgtatgct gacagcttty tgatcagagg agatgaagtg1981 cggcagattg ctcctggcca gacaggcaag attgctgact acaactacaa gctgcctgat2041 gacttcacag gctgtgtcat cgcctggaac agcaacaacc tggacagcaa ggtgggcggc2101 aactacaact acctgtacag acttttcagg aagagcaacc tgaagccttt tgaaagagac2161 atctccacag agatctacca ggctggcagc acaccctgca atggtgtgga aggcttcaac2221 tgctacttcc ctctgcagag ctacggcttc cagccaacaa atggcgtggg ctaccagcct2281 tacagagtgg tggtgctgtc ctttgagctg ctgcacgccc ctgccacagt gtgtggcccc2341 aagaagagca ccaacctggt gaagaacaaa tgtgtgaact tcaatttcaa tggcctgaca2401 ggcacaggag tgctgacaga gagcaacaag aagtttcttc ctttccagca gtttggaaga2461 gacattgctg acaccacaga tgctgtgaga gatcctcaga ccctggagat cctggatatc2521 acaccctgct cctttggagg agtttctgtc atcacacctg gcaccaatac cagcaaccaa2581 gtggctgtgc tgtaccaaga tgtgaattgc acagaagtgc ctgtggccat ccacgctgac2641 cagctgacac ccacctggag agtgtacagc acaggcagca atgttttcca gacaagagct2701 ggctgcctga ttggagcaga gcacgtgaac aacagctatg aatgtgacat ccctattgga2761 gctggcatct gtgccagcta ccagacccaa accaacagcc caagaagagc cagatctgtg2821 gccagccaga gcatcatcgc ctacaccatg agcctgggag ctgagaactc tgtggcctac2881 agcaacaaca gcatcgccat ccccaccaac ttcaccatct ctgtgaccac agagatcctg2941 cctgtgtcca tgaccaagac atctgtggac tgcaccatgt acatctgtgg agacagcaca3001 gaatgcagca acctgctgct gcagtacggc tccttctgca cccagctgaa cagagccctg3061 acaggcatcg ctgtggagca ggacaagaac acacaggaag tctttgccca ggtgaagcag3121 atctacaaaa caccacccat caaggacttt ggaggcttca atttctccca aatcctgcct3181 gaccccagca agccttccaa gagaagcttc attgaagacc tgctgttcaa caaagtgacc3241 ctggctgatg ctggcttcat caagcagtat ggagactgcc tcggagacat tgctgccaga3301 gacctgatct gtgcccagaa gtttaatggc ctgactgtgc tgcctcctct gctgacagat3361 gaaatgatcg cccagtacac atctgccctg ctggctggca ccatcaccag tggctggaca3421 tttggagctg gagctgccct gcagatccct tttgccatgc agatggccta cagatttaat3481 ggcatcggcg tgacccagaa cgtgctgtac gagaaccaga agctgatcgc caaccagttc3541 aactctgcca tcggcaagat ccaggacagc ctgagcagca cagcctctgc cctgggcaag3601 ctgcaggatg tggtgaacca aaacgcccag gccctgaaca ccctggtgaa gcagctgagc3661 agcaactttg gagccatctc ctctgtgctg aatgacatcc tgagccggct ggacaaggtg3721 gaagcagaag tccagatcga cagactcatc acaggccgcc tgcagagcct gcagacctac3781 gtgacccagc agctgatcag agctgctgag atccgggcct ctgccaacct ggctgccacc3841 aagatgtcag aatgtgtgct gggccagagc aaaagagtgg acttctgtgg caaaggctac3901 cacctgatgt ccttccctca gtctgctcct cacggcgtgg tgttcctgca cgtgacctac3961 gtgcctgccc aggagaagaa cttcaccaca gctcctgcca tctgccacga tggcaaggcc4021 cacttcccaa gagaaggtgt ctttgtgtcc aatggcaccc actggttcgt gacccagaga4081 aacttctacg agcctcagat catcaccaca gacaacacat ttgtgtctgg caactgtgat4141 gtggtcatcg gcatcgtgaa caacacagtt tatgaccctc tgcagcctga gctggacagc4201 ttcaaagaag agctggacaa gtacttcaag aaccacacat ctccagatgt ggacctggga4261 gacatctctg gcatcaatgc ctctgtggtg aacatccaga aggaaattga caggctgaac4321 gaagtggcca agaacctgaa cgaaagcctc atcgacctgc aggagctggg caagtacgag4381 cagtacatca agtggccttg gtacatctgg ctgggcttca tcgctggcct catcgccatc4441 gtgatggtga ccatcatgct gtgctgcatg accagctgct gctcttgcct gaagggctgc4501 tgcagctgtg gcagctgctg caagtttgat gaagatgact ctgagcctgt gctgaagggc4561 gtgaagctgc actacacatg ataactcgag tctagagggc ccgtttaaac ccgctgatca4621 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc4681 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg4741 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg4801 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctact4861 gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg4921 ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca4981 ggggatcaag ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg5041 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac5101 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg5161 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc5221 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg5281 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc5341 accttgctcc tgccgagaaa gtatccatca tcgctgatgc aatgcggcgg ctgcatacgc5401 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta5461 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg5521 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg5581 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat5641 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc5701 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta5761 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa5821 ttattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt5881 cacaccgcat caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt5941 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa6001 taatagcacg tcctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt6061 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc6121 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg6181 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact6241 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg6301 tagccgtagt taggccacca cttcaagaac tctgtagcac cccctacata cctcgctctg6361 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac6421 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca6481 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga6541 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc6601 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct6661 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg6781 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct6721 tttgctcaca tgttcttSARS-CoV-2 Outlier Spike Antigen amino acid insert sequenceof pGX9503 (SEQ ID NO: 4) (IgE leader sequence underlined):   1 MDWTWILFLV AAATRVHSSQ CVNLTTRTQL PPAYTNSFTR GVYYPDKVFR SSVLHSTQDL  61 FLPFFSNVTW FHAIHVSGTN GTKRFDNPVL PFNDGVYFAS TEKSNIIRGW IFGTTLDSKT 121 QSLLIVNNAT NVVIKVCEFQ FCNDPFLGVY YHKNNKSWME SEFRVYSSAN NCTFEYVSQP 181 FLMDLEGKQG NFKNLREFVF KNIDGYFKIY SKHTPINLVR DLPQGFSALE PLVDLPIGIN 241 ITRFQTLLAL HRSYLTPGDS SSGWTAGAAA YYVGYLQPRT FLLKYNENGT ITVAVACALD 301 PLSETKCTLK SFTVEKGIYQ TSNFRVQPTE SIVRFPNITN LCPFGEVENA TRFASVYAWN 361 RKRISNCVAD YSVLYNSASF STFKCYGVSP TKLNDLCFTN VYADSFVIRG DEVRQIAPGQ 421 TGKIADYNYK LPDDFTGCVI AWNSNNLDSK VGGNYNYLYR LFRKSNLKPF ERDISTEIYQ 481 AGSTPCNGVE GENCYFPLQS YGFQPTNGVG YQPYRVVVLS FELLHAPATV CGPKKSTNLV 541 KNKCVNFNFN GLTGTGVLTE SNKKFLPFQQ FGRDIADTTD AVRDPQTLEI LDITPCSFGG 601 VSVITPGANT SNQVTVLYQD VNCTEVPVAI HADQLTPTWR VYSTGSNVFK TRAGCLIGAE 661 HVNNSYECDI PIGAGICASY QTQTNSPRRA RSTASQSIIA YTMSLGAENS VAYSNNSIVI 721 PTNFTISVTT EILPVSMTKT SVDCTMYICS DSTECSNPLL QYGSFCTQLN RALTGIAVEQ 781 DKNTQEVFAQ VKQIYKTPPI KDFGGFNFSQ ILPDPSKPSK RSFIEDLLFN KVTLADAGFI 841 KQYGDCLGDI AARDLICAQK FNGLTVLPPL LTDEMIAQYT SALLAGTITS GWTFGAGAAL 901 QIPFAMQMAY RENGIRVTQN VLYENQKLIA NQFNSAIGKI QDSLSSTASA LGKLQDVVNQ 961 NAQALNTLVK QLSSTFSTIS SVLNDILSRL DKVEAEVQID RLITGRLQSL QTYVTQQLIR1021 AAEIRASANL KATKMSECVL GQSKRVDFCG KGYHLMSFPQ SAPHGVVFLH VTYVPAQEKN1081 FTTAPATCHD GKAHFPREGV FVSNGTHWFV TQRNFDEPQI ITTDNTFVSG NCDVVIGIVN1141 NTVYDPLQPE LDSFKEELDK YFKNHTSPDV DLGDISGINA SVVNIQKEID RLNEVAKNLN1201 ESLIDLQELG KYEQYIKWPW YIWLGFIAGL IAIVMVTIML CCMTSCCSCL KGCCSCGSCC1261 KFDEDDSEPV LKGVKLHYT DNA insert sequence of pGX9503 (SEQ ID NO: 5)(IgE leader sequence underlined):   1 ATGGATTGGA CCTGGATTCT TTTTCTCGTT GCAGCTGCTA CACGCGTTCA TAGCAGCCAG  61 TGTGTGAACC TGACCACCAG AACACAGCTG CCTCCTGCCT ACACCAACAG CTTCACCAGA 121 GGAGTCTACT ACCCAGACAA GGTGTTCAGA AGCTCTGTGC TGCACAGCAC CCAGGACCTC 181 TTCCTGCCTT TCTTCAGCAA CGTGACCTGG TTCCACGCCA TCCACGTGTC TGGCACCAAC 241 GGCACCAAGA GATTTGACAA CCCTGTGCTG CCTTTCAATG ATGGTGTGTA CTTTGCCAGC 301 ACAGAGAAGA GCAACATCAT CCGAGGCTGG ATCTTTGGCA CCACCCTGGA CAGCAAAACA 361 CAGAGCCTGC TGATCGTGAA TAATGCCACC AACGTGGTCA TCAAGGTGTG TGAGTTCCAG 421 TTCTGCAATG ACCCTTTCCT GGGCGTGTAC TACCACAAGA ACAACAAGTC CTGGATGGAG 481 TCTGAGTTCC GAGTGTACAG CTCTGCCAAC AACTGCACAT TTGAATATGT GTCCCAGCCT 541 TTCCTGATGG ACCTGGAGGG CAAGCAGGGC AATTTCAAGA ACCTGAGAGA ATTTGTGTTC 601 AAGAACATCG ATGGCTACTT CAAGATCTAC AGCAAGCACA CACCCATCAA CCTGGTGAGA 661 GATCTTCCTC AGGGCTTCTC TGCCCTGGAG CCTCTGGTGG ACCTGCCCAT CGGCATCAAC 721 ATCACCCGCT TTCAGACCCT GCTGGCCCTG CACAGAAGCT ACCTGACCCC AGGAGACAGC 781 AGCAGCGGCT GGACAGCTGG AGCTGCTGCC TACTACGTGG GCTACCTGCA GCCAAGAACC 841 TTCCTGCTGA AGTACAACGA AAATGGCACC ATCACTGTGG CTGTGGCCTG TGCCCTGGAC 901 CCTCTTTCTG AGACCAAGTG CACCCTGAAG TCCTTCACAG TGGAGAAAGG CATCTACCAG 961 ACCAGCAACT TCAGAGTTCA GCCAACAGAG AGCATCGTGA GATTTCCAAA CATCACCAAC1021 CTGTGTCCTT TTGGAGAAGT CTTCAATGCC ACCAGATTTG CTTCTGTGTA CGCCTGGAAC1081 AGAAAAAGAA TCAGCAACTG TGTGGCTGAC TACTCTGTGC TGTACAACTC TGCCTCCTTC1141 TCCACCTTCA AGTGCTACGG TGTGTCTCCT ACCAAGCTGA ATGACCTGTG CTTCACCAAC1201 GTGTATGCTG ACAGCTTTGT CATCAGAGGA GATGAAGTGC GGCAGATCGC CCCTGGCCAG1261 ACAGGCAAGA TTGCTGACTA CAACTACAAG CTGCCTGATG ACTTCACAGG CTGTGTCATC1321 GCCTGGAACA GCAACAACCT GGACAGCAAG GTGGGCGGCA ACTACAACTA CCTGTACAGA1381 CTTTTCAGGA AGAGCAACCT GAAGCCTTTT GAAAGAGACA TCTCCACAGA GATCTACCAG1441 GCTGGCAGCA CACCCTGCAA TGGAGTGGAA GGCTTCAACT GCTACTTCCC TCTGCAGAGC1501 TACGGCTTCC AGCCCACCAA TGGCGTGGGC TACCAGCCTT ACAGAGTGGT GGTGCTGTCC1561 TTTGAGCTGC TGCACGCCCC TGCCACAGTG TGTGGCCCCA AGAAGAGCAC CAACCTGGTG1621 AAGAACAAAT GTGTGAACTT CAATTTCAAT GGCCTGACAG GCACAGGAGT GCTGACAGAG1681 AGCAACAAGA AGTTCCTGCC TTTCCAGCAG TTTGGAAGAG ACATTGCTGA CACCACAGAT1741 GCTGTGAGAG ATCCTCAGAC CCTGGAGATC CTGGACATCA CACCCTGCTC CTTTGGAGGA1801 GTTTCTGTCA TCACACCTGG AGCCAACACC AGCAACCAAG TGACAGTGCT GTACCAAGAT1861 GTGAACTGCA CAGAAGTTCC TGTGGCCATC CACGCTGACC AGCTGACCCC AACCTGGAGA1921 GTCTACAGCA CAGGCAGCAA CGTGTTTAAA ACAAGAGCTG GCTGCCTGAT TGGAGCAGAG1981 CACGTGAACA ACAGCTATGA ATGTGACATC CCTATTGGAG CTGGCATCTG TGCCAGCTAC2041 CAGACCCAAA CCAACAGCCC AAGAAGAGCC AGGAGCACAG CCAGCCAGAG CATCATCGCC2101 TACACCATGA GCCTGGGAGC AGAGAACTCT GTGGCCTACA GCAACAACAG CATCGTCATC2161 CCCACCAACT TCACCATCTC TGTGACCACA GAGATCCTGC CTGTGTCCAT GACCAAGACA2221 TCTGTGGACT GCACCATGTA CATCTGCAGT GACAGCACAG AATGCAGCAA CCCTCTGCTG2281 CAGTACGGCT CCTTCTGCAC CCAGCTGAAC AGAGCCCTGA CAGGCATCGC TGTGGAGCAG2341 GACAAGAACA CACAGGAAGT GTTTGCCCAG GTGAAGCAGA TCTACAAAAC ACCACCCATC2401 AAGGACTTTG GAGGCTTCAA CTTCTCCCAG ATCCTGCCTG ACCCCAGCAA GCCCAGCAAG2461 AGAAGCTTCA TTGAAGACCT GCTGTTCAAC AAAGTGACCC TGGCTGATGC TGGCTTCATC2521 AAACAATATG GAGACTGCCT GGGAGACATT GCTGCCAGAG ACCTGATCTG TGCCCAGAAG2581 TTTAATGGCC TGACTGTGCT GCCTCCTCTG CTGACAGATG AAATGATCGC CCAGTACACA2641 TCTGCCCTGC TGGCTGGCAC CATCACATCT GGCTGGACAT TTGGAGCTGG AGCTGCCCTG2701 CAGATCCCTT TTGCCATGCA GATGGCCTAC AGATTTAATG GCATCAGAGT GACCCAGAAC2761 GTGCTGTATG AAAACCAGAA GCTGATCGCC AACCAGTTCA ACTCTGCCAT CGGCAAGATC2821 CAGGACAGCC TGAGCAGCAC AGCCTCTGCC CTGGGCAAGC TGCAGGATGT GGTGAACCAA2881 AATGCCCAGG CCCTGAACAC CCTGGTGAAG CAGCTGAGCA GCACCTTCTC CACCATCTCC2941 AGCGTGCTGA ATGACATCCT GAGCCGGCTG GACAAGGTGG AAGCTGAGGT GCAGATCGAC3001 AGACTCATCA CAGGCCGGCT GCAGAGCCTG CAGACCTACG TGACCCAGCA GCTGATCAGA3001 AGACTCATCA CAGGCCGGCT GCAGAGCCTG CAGACCTACG TGACCCAGCA GCTGATCAGA3061 GCTGCTGAGA TCAGAGCTTC TGCCAACCTG AAGGCCACCA AGATGTCAGA ATGTGTGCTG3121 GGCCAGAGCA AGAGAGTGGA CTTCTGTGGC AAAGGCTACC ACCTGATGTC CTTCCCTCAG3181 TCTGCTCCTC ACGGCGTGGT GTTCCTGCAC GTGACCTACG TGCCTGCCCA GGAGAAGAAC3241 TTCACCACAG CTCCTGCCAC CTGCCACGAT GGCAAAGCCC ACTTCCCAAG AGAAGGCGTC3301 TTTGTGTCCA ATGGCACCCA CTGGTTCGTG ACCCAGAGAA ACTTTGATGA GCCTCAGATC3361 ATCACCACAG ACAACACATT TGTTTCTGGC AACTGTGATG TGGTCATCGG CATCGTGAAC3421 AACACAGTTT ATGACCCTCT GCAGCCTGAG CTGGACAGCT TCAAAGAAGA GCTGGACAAG3481 TACTTCAAGA ACCACACATC TCCAGATGTG GACCTGGGAG ACATCTCTGG CATCAATGCC3541 TCTGTGGTGA ACATCCAGAA GGAAATTGAC AGGCTGAACG AAGTGGCCAA GAACCTGAAC3601 GAAAGCCTCA TCGACCTGCA GGAGCTGGGC AAGTACGAGC AGTACATCAA GTGGCCTTGG3661 TACATCTGGC TGGGCTTCAT TGCTGGCCTC ATCGCCATCG TGATGGTGAC CATCATGCTG3721 TGCTGCATGA CCAGCTGCTG CTCTTGCCTG AAGGGCTGCT GCAGCTGTGG CAGCTGCTGC3781 AAGTTTGATG AAGATGACTC TGAGCCTGTG CTGAAGGGCG TGAAGCTGCA CTACACASingle strand DNA sequence of pGX9503 (SEQ ID NO: 6):   1 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta  61 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 121 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 181 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 241 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 301 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 361 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 421 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 481 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 541 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 601 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 661 aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 721 accgagctcg gatccgccac catggattgg acctggattc tttttctcgt tgcagctgct 781 acacgcgttc atagcagcca gtgtgtgaac ctgaccacca gaacacagct gcctcctgcc 841 tacaccaaca gcttcaccag aggagtctac tacccagaca aggtgttcag aagctctgtg 901 ctgcacagca cccaggacct cttcctgcct ttcttcagca acgtgacctg gttccacgcc 961 atccacgtgt ctggcaccaa cggcaccaag agatttgaca accctgtgct gcctttcaat1021 gatggtgtgt actttgccag cacagagaag agcaacatca tccgaggctg gatctttggc1081 accaccctgg acagcaaaac acagagcctg ctgatcgtga ataatgccac caacgtggtc1141 atcaaggtgt gtgagttcca gttctgcaat gaccctttcc tgggcgtgta ctaccacaag1201 aacaacaagt cctggatgga gtctgagttc cgagtgtaca gctctgccaa caactgcaca1261 tttgaatatg tgtcccagcc tttcctgatg gacctggagg gcaagcaggg caatttcaag1321 aacctgagag aatttgtgtt caagaacatc gatggctact tcaagatcta cagcaagcac1381 acacccatca acctggtgag agatcttcct cagggcttct ctgccctgga gcctctggtg1441 gacctgccca tcggcatcaa catcacccgc tttcagaccc tgctggccct gcacagaagc1501 tacctgaccc caggagacag cagcagcggc tggacagctg gagctgctgc ctactacgtg1561 ggctacctgc agccaagaac cttcctgctg aagtacaacg aaaatggcac catcactgtg1621 gctgtggcct gtgccctgga ccctctttct gagaccaagt gcaccctgaa gtccttcaca1681 gtggagaaag gcatctacca gaccagcaac ttcagagttc agccaacaga gagcatcgtg1741 agatttccaa acatcaccaa cctgtgtcct tttggagaag tcttcaatgc caccagattt1801 gcttctgtgt acgcctggaa cagaaaaaga atcagcaact gtgtggctga ctactctgtg1861 ctgtacaact ctgcctcctt ctccaccttc aagtgctacg gtgtgtctcc taccaagctg1921 aatgacctgt gcttcaccaa cgtgtatgct gacagctttg tcatcagagg agatgaagtg1981 cggcagatcg cccctggcca gacaggcaag attgctgact acaactacaa gctgcctgat2041 gacttcacag gctgtgtcat cgcctggaac agcaacaacc tggacagcaa ggtgggcggc2101 aactacaact acctgtacag acttttcagg aagagcaacc tgaagccttt tgaaagagac2161 atctccacag agatctacca ggctggcagc acaccctgca atggagtgga aggcttcaac2221 tgctacttcc ctctgcagag ctacggcttc cagcccacca atggcgtggg ctaccagcct2281 tacagagtgg tggtgctgtc ctttgagctg ctgcacgccc ctgccacagt gtgtggcccc2341 aagaagagca ccaacctggt gaagaacaaa tgtgtgaact tcaatttcaa tggcctgaca2401 ggcacaggag tgctgacaga gagcaacaag aagttcctgc ctttccagca gtttggaaga2461 gacattgctg acaccacaga tgctgtgaga gatcctcaga ccctggagat cctggacatc2521 acaccctgct cctttggagg agtttctgtc atcacacctg gagccaacac cagcaaccaa2581 gtgacagtgc tgtaccaaga tgtgaactgc acagaagttc ctgtggccat ccacgctgac2641 cagctgaccc caacctggag agtctacagc acaggcagca acgtgtttaa aacaagagct2701 ggctgcctga ttggagcaga gcacgtgaac aacagctatg aatgtgacat ccctattgga2761 gctggcatct gtgccagcta ccagacccaa accaacagcc caagaagagc caggagcaca2821 gccagccaga gcatcatcgc ctacaccatg agcctgggag cagagaactc tgtggcctac2881 agcaacaaca gcatcgtcat ccccaccaac ttcaccatct ctgtgaccac agagatcctg2941 cctgtgtcca tgaccaagac atctgtggac tgcaccatgt acatctgcag tgacagcaca3001 gaatgcagca accctctgct gcagtacggc tccttctgca cccagctgaa cagagccctg3061 acaggcatcg ctgtggagca ggacaagaac acacaggaag tgtttgccca ggtgaagcag3121 atctacaaaa caccacccat caaggacttt ggaggcttca acttctccca gatcctgcct3181 gaccccagca agcccagcaa gagaagcttc attgaagacc tgctgttcaa caaagtgacc3241 ctggctgatg ctggcttcat caaacaatat ggagactgcc tcggagacat tgctgccaga3301 gacctgatct gtgcccagaa gtttaatggc ctgactgtgc tgcctcctct gctgacagat3361 gaaatgatcg cccagtacac atctgccctg ctggctggca ccatcacatc tggctggaca3421 tttggagctg gagctgccct gcagatccct tttgccatgc agatggccta cagatttaat3481 ggcatcagag tgacccagaa cgtgctgtat gaaaaccaga agctgatcgc caaccagttc3541 aactctgcca tcggcaagat ccaggacagc ctgagcagca cagcctctgc cctgggcaag3601 ctgcaggatg tggtgaacca aaatgcccag gccctgaaca ccctggtgaa gcagctgagc3661 agcaccttct ccaccatctc cagcgtgctg aatgacatcc tgagccggct ggacaaggtg3721 gaagctgagg tccagatcga cagactcatc acaggccggc tgcagagcct gcagacctac3781 gtgacccagc agctgatcag agctgctgag atcagagctt ctgccaacct gaaggccacc3841 aagatgtcag aatgtgtgct gggccagagc aagagagtgg acttctgtgg caaaggctac3901 cacctgatgt ccttccctca gtctgctcct cacggcgtgg tgttcctgca cgtgacctac3961 gtgcctgccc aggagaagaa cttcaccaca gctcctgcca cctgccacga tggcaaagcc4021 cacttcccaa gagaaggcgt ctttgtgtcc aatggcaccc actggttcgt gacccagaga4081 aactttgatg agcctcagat catcaccaca gacaacacat ttgtttctgg caactgtgat4141 gtggtcatcg gcatcgtgaa caacacagtt tatgaccctc tgcagcctga gctggacagc4201 ttcaaagaag agctggacaa gtacttcaag aaccacacat ctccagatgt ggacctggga4261 gacatctctg gcatcaatgc ctctgtggtg aacatccaga aggaaattga caggctgaac4321 gaagtggcca agaacctgaa cgaaagcctc atcgacctgc aggagctggg caagtacgag4381 cagtacatca agtggccttg gtacatctgg ctgggcttca ttgctggcct catcgccatc4441 gtgatggtga ccatcatgct gtgctgcatg accagctgct gctcttgcct gaagggctgc4501 tgcagctgtg gcagctgctg caagtttgat gaagatgact ctgagcctgt gctgaagggc4561 gtgaagctgc actacacatg ataactcgag tctagagggc ccgtttaaac ccgctgatca4621 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc4681 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg4741 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg4801 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctact4861 gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg4921 ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca4981 ggggatcaag ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg5041 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac5101 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg5161 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc5221 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg5281 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc5341 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc5401 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta5461 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg5521 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg5581 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat5641 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc5701 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta5761 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa5821 ttattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt5881 cacaccgcat caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt5941 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa6001 taatagcacg tcctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt6061 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc6121 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg6181 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact6241 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg6301 tagccgtagt taggccacca cttcaagaac tctgtagcac cccctacata cctcgctctg6361 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac6421 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca6481 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga6541 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc6601 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct6661 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg6781 Agcctatgga Aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct6721 tttgctcaca tgttctt SEQ ID NO: 7 pGX9501 Forward primerCAGGACAAGAACACACAGGAA SEQ ID NO: 8 pGX9501 Reverse primerCAGGCAGGATTTGGGAGAAA SEQ ID NO: 9 pGX9501 Probe ACCCATCAAGGACTTTGGAGGSEQ ID NO: 10 pGX9503 Forward primer AGGACAAGAACACACAGGAAG;SEQ ID NO: 11 pGX9503 Reverse primer CAGGATCTGGGAGAAGTTGAAGSEQ ID NO: 12 pGX9503 Probe ACACCACCCATCAAGGACTTTGGASEQ ID NO: 13 β-actin Forward primer GTGACGTGGACATCCGTAAASEQ ID NO: 14 β-actin Reverse primer CAGGGCAGTAATCTCCTTCTGSEQ ID NO: 15 β-actin Probe TACCCTGGCATTGCTGACAGGATG SEQ ID NO: 16PHGVVFLHV SEQ ID NO: 17 VVFLHVTVYV SEQ ID NO: 18: 2019-nCoV N1-F5′-GACCCCAAAATCAGCGAAAT-3′ SEQ ID NO: 19: 2019-nCoV N1-R5′-TCTGGTTACTGCCAGTTGAATCTG-3′ SEQ ID NO: 20: 2019-nCoV N1-P5′-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3′SEQ ID NO: 21: 2019-nCoV sgE-forward 5′CGATCTCTTGTAGATCTGTTCTC 3′SEQ ID NO: 22: 2019-nCoV sgE-reverse 5′ATATTGCAGCAGTACGCACACA 3′SEQ ID NO: 23: 2019-nCoV sgE-probe5′FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1 3′

What is claimed:
 1. A nucleic acid molecule encoding a Severe AcuteRespiratory Syndrome coronavirus 2 (SARS-CoV-2) spike antigen, thenucleic acid molecule comprising: a nucleic acid sequence having atleast about 90% identity over an entire length of the nucleic acidsequence set forth from nucleotide 55 to nucleotide 3837 of SEQ ID NO:2, a nucleic acid sequence having at least about 90% identity over anentire length of SEQ ID NO: 2, the nucleic acid sequence from nucleotide55 to nucleotide 3837 of SEQ ID NO: 2, the nucleic acid sequence of SEQID NO: 2, a nucleic acid sequence having at least about 90% identityover an entire length of SEQ ID NO: 3, the nucleic acid sequence of SEQID NO: 3, a nucleic acid sequence having at least about 90% identityover an entire length of the nucleic acid sequence from nucleotide 55 tonucleotide 3837 of SEQ ID NO: 5, a nucleic acid sequence having at leastabout 90% identity over an entire length of SEQ ID NO: 5, the nucleicacid sequence from nucleotide 55 to nucleotide 3837 of SEQ ID NO: 5, thenucleic acid sequence of SEQ ID NO: 5, a nucleic acid sequence having atleast about 90% identity over an entire length of SEQ ID NO: 6, or thenucleic acid sequence of SEQ ID NO:
 6. 2. An expression vectorcomprising a nucleic acid molecule encoding a Severe Acute RespiratorySyndrome coronavirus 2 (SARS-CoV-2) spike antigen, the nucleic acidmolecule comprising: a nucleic acid sequence having at least about 90%identity over an entire length of the nucleic acid sequence set forthfrom nucleotide 55 to nucleotide 3837 of SEQ ID NO: 2, a nucleic acidsequence having at least about 90% identity over an entire length of SEQID NO: 2, the nucleic acid sequence from nucleotide 55 to nucleotide3837 of SEQ ID NO: 2, the nucleic acid sequence of SEQ ID NO: 2, anucleic acid sequence having at least about 90% identity over an entirelength of SEQ ID NO: 3, the nucleic acid sequence of SEQ ID NO: 3, anucleic acid sequence having at least about 90% identity over an entirelength of the nucleic acid sequence from nucleotide 55 to nucleotide3837 of SEQ ID NO: 5, a nucleic acid sequence having at least about 90%identity over an entire length of SEQ ID NO: 5, the nucleic acidsequence from nucleotide 55 to nucleotide 3837 of SEQ ID NO: 5, thenucleic acid sequence of SEQ ID NO: 5, a nucleic acid sequence having atleast about 90% identity over an entire length of SEQ ID NO: 6, or thenucleic acid sequence of SEQ ID NO:
 6. 3. An immunogenic compositioncomprising an effective amount of an expression vector and apharmaceutically acceptable excipient, wherein the expression vectorcomprises a nucleic acid molecule encoding a Severe Acute RespiratorySyndrome coronavirus 2 (SARS-CoV-2) spike antigen, the nucleic acidmolecule comprising: a nucleic acid sequence having at least about 90%identity over an entire length of the nucleic acid sequence set forthfrom nucleotide 55 to nucleotide 3837 of SEQ ID NO: 2, a nucleic acidsequence having at least about 90% identity over an entire length of SEQID NO: 2, the nucleic acid sequence from nucleotide 55 to nucleotide3837 of SEQ ID NO: 2, the nucleic acid sequence of SEQ ID NO: 2, anucleic acid sequence having at least about 90% identity over an entirelength of SEQ ID NO: 3, the nucleic acid sequence of SEQ ID NO: 3, anucleic acid sequence having at least about 90% identity over an entirelength of the nucleic acid sequence from nucleotide 55 to nucleotide3837 of SEQ ID NO: 5, a nucleic acid sequence having at least about 90%identity over an entire length of SEQ ID NO: 5, the nucleic acidsequence from nucleotide 55 to nucleotide 3837 of SEQ ID NO: 5, thenucleic acid sequence of SEQ ID NO: 5, a nucleic acid sequence having atleast about 90% identity over an entire length of SEQ ID NO: 6, or thenucleic acid sequence of SEQ ID NO:
 6. 4. The immunogenic compositionaccording to claim 3, wherein the pharmaceutically acceptable excipientcomprises a buffer.
 5. The immunogenic composition according to claim 4,wherein the buffer is saline-sodium citrate buffer.
 6. The immunogeniccomposition of claim 5, wherein the composition comprises 10 mg of thevector per milliliter of saline-sodium citrate buffer.
 7. Theimmunogenic composition according to claim 3, further comprising anadjuvant.
 8. A vaccine for the prevention or treatment of Severe AcuteRespiratory Syndrome coronavirus 2 (SARS-CoV-2) infection comprising aneffective amount of the nucleic acid molecule of claim 1 and apharmaceutically acceptable excipient.
 9. The vaccine according to claim8, further comprising an adjuvant.
 10. A method of inducing an immuneresponse against Severe Acute Respiratory Syndrome coronavirus 2(SARS-CoV-2) in a subject in need thereof, the method comprisingadministering an effective amount of an immunogenic composition to thesubject, wherein the immunogenic composition comprises an expressionvector comprising a nucleic acid molecule encoding a SARS-CoV-2 spikeantigen, the nucleic acid molecule comprising: a nucleic acid sequencehaving at least about 90% identity over an entire length of the nucleicacid sequence set forth from nucleotide 55 to nucleotide 3837 of SEQ IDNO: 2, a nucleic acid sequence having at least about 90% identity overan entire length of SEQ ID NO: 2, the nucleic acid sequence fromnucleotide 55 to nucleotide 3837 of SEQ ID NO: 2, the nucleic acidsequence of SEQ ID NO: 2, a nucleic acid sequence having at least about90% identity over an entire length of SEQ ID NO: 3, the nucleic acidsequence of SEQ ID NO: 3, a nucleic acid sequence having at least about90% identity over an entire length of the nucleic acid sequence fromnucleotide 55 to nucleotide 3837 of SEQ ID NO: 5, a nucleic acidsequence having at least about 90% identity over an entire length of SEQID NO: 5, the nucleic acid sequence from nucleotide 55 to nucleotide3837 of SEQ ID NO: 5, the nucleic acid sequence of SEQ ID NO: 5, anucleic acid sequence having at least about 90% identity over an entirelength of SEQ ID NO: 6, or the nucleic acid sequence of SEQ ID NO: 6,and a pharmaceutically acceptable excipient.
 11. The method of claim 10,wherein administering comprises at least one of electroporation andparenteral administration.
 12. The method of claim 10, whereinadministering comprises parenteral administration followed byelectroporation.
 13. The method of claim 10, further comprisingadministering to the subject at least one additional agent for thetreatment of SARS-CoV-2 infection or the treatment or prevention of adisease or disorder associated with SARS-CoV-2 infection.
 14. The methodof claim 13 wherein the immunogenic composition is administered to thesubject before, concurrently with, or after the additional agent.
 15. Amethod of protecting a subject in need thereof from infection withSevere Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), the methodcomprising administering to the subject an effective amount of thenucleic acid molecule of claim
 1. 16. A method of protecting a subjectin need thereof from a disease or disorder associated with infectionwith Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), themethod comprising administering to the subject an effective amount ofthe nucleic acid molecule of claim
 1. 17. A method of treating a subjectin need thereof against Severe Acute Respiratory Syndrome coronavirus 2(SARS-CoV-2), the method comprising administering to the subject aneffective amount of the nucleic acid molecule of claim
 1. 18. A methodof detecting a persistent cellular immune response in a subject, themethod comprising the steps of: administering an immunogenic compositionfor inducing an immune response against a SARS-CoV-2 antigen to asubject in need thereof; isolating peripheral mononuclear cells (PBMCs)from the subject; stimulating the isolated PBMCs with a SARS-CoV-2 spikeantigen comprising an amino acid sequence selected from the groupconsisting of an amino acid sequence having at least about 90% identityover an entire length of residues 19 to 1279 of SEQ ID NO: 1, the aminoacid sequence set forth in residues 19 to 1279 of SEQ ID NO: 1, an aminoacid sequence having at least about 90% identity over an entire lengthof SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 1, an amino acidsequence having at least about 90% identity over an entire length ofresidues 19 to 1279 of SEQ ID NO: 4, an amino acid sequence having atleast about 90% identity over an entire length of SEQ ID NO: 4, theamino acid sequence set forth in residues 19 to 1279 of SEQ ID NO: 4,the amino acid sequence of SEQ ID NO: 4, and a fragment thereofcomprising at least 20 amino acids; and detecting at least one of thenumber of cytokine expressing cells and the level of cytokineexpression.
 19. The method of claim 18, wherein the step of detecting atleast one of the number of cytokine expressing cells and the level ofcytokine expression is performed using an assay selected from the groupconsisting of Enzyme-linked immunospot (ELISpot) and IntracellularCytokine Staining (ICS) analysis using flow cytometry.
 20. The method ofclaim 18, wherein the subject is administered an immunogenic compositioncomprising a nucleic acid molecule, the nucleic acid moleculecomprising: a nucleic acid sequence having at least about 90% identityover an entire length of the nucleic acid sequence set forth fromnucleotide 55 to nucleotide 3837 of SEQ ID NO: 2, a nucleic acidsequence having at least about 90% identity over an entire length of SEQID NO: 2, the nucleic acid sequence from nucleotide 55 to nucleotide3837 of SEQ ID NO: 2, the nucleic acid sequence of SEQ ID NO: 2, anucleic acid sequence having at least about 90% identity over an entirelength of SEQ ID NO: 3, the nucleic acid sequence of SEQ ID NO: 3, anucleic acid sequence having at least about 90% identity over an entirelength of the nucleic acid sequence from nucleotide 55 to nucleotide3837 of SEQ ID NO: 5, a nucleic acid sequence having at least about 90%identity over an entire length of SEQ ID NO: 5, the nucleic acidsequence from nucleotide 55 to nucleotide 3837 of SEQ ID NO: 5, thenucleic acid sequence of SEQ ID NO: 5, a nucleic acid sequence having atleast about 90% identity over an entire length of SEQ ID NO: 6, or thenucleic acid sequence of SEQ ID NO: 6.